WO2005081905A2 - Systemes et methodes d'expression clonale dans des plantes - Google Patents

Systemes et methodes d'expression clonale dans des plantes Download PDF

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Publication number
WO2005081905A2
WO2005081905A2 PCT/US2005/005409 US2005005409W WO2005081905A2 WO 2005081905 A2 WO2005081905 A2 WO 2005081905A2 US 2005005409 W US2005005409 W US 2005005409W WO 2005081905 A2 WO2005081905 A2 WO 2005081905A2
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Prior art keywords
clonal
plant
polynucleotide
root
viral vector
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PCT/US2005/005409
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English (en)
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WO2005081905A3 (fr
Inventor
Vidadi Yusibov
Marina Skarjinskaia
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Fraunhofer Usa Inc.
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Priority to CN2005800055022A priority Critical patent/CN1922306B/zh
Priority to AU2005216133A priority patent/AU2005216133A1/en
Priority to CA002555230A priority patent/CA2555230A1/fr
Priority to EP05723389.2A priority patent/EP1769068B1/fr
Priority to ES05723389.2T priority patent/ES2532609T3/es
Publication of WO2005081905A2 publication Critical patent/WO2005081905A2/fr
Publication of WO2005081905A3 publication Critical patent/WO2005081905A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology

Definitions

  • plants have been increasingly used as a host system for the expression of recombinant proteins.
  • Such expression can be accomplished, for example, either by integrating the gene of interest into a plant genome, to create a transgenic plant that stably expresses the desired protein, or by introducing the gene of interest into a plant vector that can be introduced into, and transiently maintained in, plant cells.
  • Niral vector systems have proven to be particularly useful.
  • viruses may infect non- target plants, potentially posing significant environmental risks.
  • many available engineered plant viruses do not express inserted genes at desired levels, and/or in desired target plants or tissues.
  • virus stability can be problematic.
  • plant expression systems that would allow for greater flexibility and control.
  • the present invention encompasses the recognition that the availability of clonal expression systems for plants would offer a number of significant advantages.
  • the invention provides methods and reagents for generating a variety of clonal entities derived from plants. These clonal entities include clonal root lines, clonal root cell lines, clonal plant cell lines, and clonal plants.
  • the invention further provides methods and reagents for expression of polynucleotide and polypeptide products in clonal cell lines derived from various plant tissues (e.g., roots, leaves), and in whole plants derived from single cells (clonal plants). The methods are based on the use of plant viral vectors of various types.
  • the invention provides a method of obtaining a clonal root line that expresses a polynucleotide of interest comprising steps of: (i) introducing a viral vector that comprises a polynucleotide of interest into a plant or portion thereof; and (ii) generating one or more clonal root lines from the plant.
  • the clonal root lines may be generated, for example, by infecting the plant or plant portion (e.g., a harvested piece of leaf) with an Agrobacterium (e.g., A. rhizogenes) that causes formation of hairy roots.
  • Clonal root lines can be screened in various ways to identify lines that maintain the virus, lines that express the polynucleotide of interest at high levels, etc.
  • the invention further provides clonal root lines, e.g., clonal root lines produced according to the inventive methods and further encompasses methods of expressing polynucleotides and producing polypeptides of interest using the clonal root lines.
  • the invention further provides a method of generating a clonal root cell line that expresses a polynucleotide of interest comprising steps of: (i) generating a clonal root line, cells of which contain a viral vector whose genome comprises a polynucleotide of interest; (ii) releasing individual cells from the clonal root line; and (iii) maintaining the cells under conditions suitable for root cell proliferation.
  • the invention provides clonal root cell lines and methods of expressing polynucleotides and producing polypeptides using the clonal root cell lines.
  • the invention provides a method of generating a clonal plant cell line that expresses a polynucleotide of interest comprising steps of: (i) generating a clonal root line, cells of which contain a viral vector whose genome comprises a polynucleotide of interest;
  • the invention further provides a method of generating a clonal plant cell line that expresses a polynucleotide of interest comprising steps of: (i) introducing a viral vector that comprises a polynucleotide of interest into cells of a plant cell line maintained in culture; and (ii) enriching for cells that contain the viral vector.
  • Enrichment may be- performed, for example, by (i) removing a portion ofthe cells from the culture; (ii) diluting the removed cells so as to reduce the cell concentration; (iii) allowing the diluted cells to proliferate; and (iv) screening for cells that contain the viral vector.
  • Clonal plant cell lines may be used for production of a polypeptide of interest.
  • the invention provides a method of generating a clonal plant that expresses a polynucleotide of interest comprising steps of: (i) generating a clonal root line, cells of which contain a viral vector whose genome comprises a polynucleotide of interest; (ii) releasing individual cells from the clonal root line; and (iii) maintaining the cells under conditions appropriate for formation of a plant.
  • the invention further provides a method of generating a clonal plant that expresses a polynucleotide of interest comprising steps of: (i) generating a clonal plant cell line, cells of which contain a viral vector whose genome comprises a polynucleotide of interest; and (ii) maintaining the cells under conditions appropriate for formation of a plant.
  • the clonal plants can express any polynucleotide of interest.
  • the clonal plants can be used for production of a polypeptide of interest.
  • Figure I presents a schematic diagram ofthe engineering of a TMN based viral construct containing a polynucleotide of interest.
  • the upper portion ofthe figure shows a diagram ofthe genomic organization of a TMN based virus construct, D4, and the lower portion shows the same construct following insertion of a polynucleotide of interest (e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target”).
  • the 126/183 kDa protein is required for replication ofthe virus.
  • the 30 l D protein is the movement protein (MP) that mediates cell-to-cell movement.
  • Arrows indicate positions of the subgenomic promoters. Transcription ofthe inserted polynucleotide is under control ofthe TMN CP subgenomic promoter.
  • the 3' portion ofthe construct includes TMN coat protein sequences and untranslated regions. These portions are optional.
  • Figure 2 presents a schematic diagram ofthe engineering of a TMN based viral construct containing a polynucleotide of interest.
  • the upper portion ofthe figure shows a schematic diagram ofthe genomic organization of a TMV based virus nnnctrn p t 0U ⁇ i- ⁇ 1n pr nnrtinn s ows the. same construct followine insertion of a polynucleotide of interest (e.g., a gene encoding liGH, GCSF, GFP, etc., indicated as "target”).
  • the 126/183 kDa protein is required for replication ofthe virus.
  • the 30 l D protein is the movement protein (MP) that mediates cell-to-cell movement.
  • MP movement protein
  • CP is the coat protein that mediates systemic spread. Arrows indicate positions ofthe subgenomic promoters. Transcription ofthe inserted polynucleotide is under control of an introduced promoter. CP expression is under control ofthe endogenous CP promoter.
  • the 3' portion ofthe construct includes TMN coat protein sequences and untranslated regions. These portions are optional.
  • Figure 3 presents a schematic diagram ofthe engineering of a TMN based viral construct containing a polynucleotide of interest and a gene encoding a marker for detection and/or selection.
  • the upper portion ofthe figure shows the genomic organization of a TMN based virus construct, D4.
  • the middle portion ofthe figure shows the same construct after insertion of a gene encoding a detectable marker (GFP) replacing the MP coding sequence.
  • the lower portion ofthe figure shows the same construct following insertion of a polynucleotide of interest (e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target”).
  • the 126/183 kDa protein is required for replication ofthe virus.
  • FIG. 4 presents a schematic diagram ofthe engineering of a TMN based viral construct containing a polynucleotide of interest and a gene encoding a marker for detection and/or selection.
  • the upper portion ofthe figure shows the genomic organization of a TMN based virus construct, D4.
  • the middle portion ofthe figure shows the same construct after insertion of a gene encoding a selectable marker (gene encoding resistance to kanamycin) replacing the MP coding sequence.
  • the lower portion ofthe figure shows the same construct following insertion of a polynucleotide of interest (e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target”).
  • the 126/183 kDa protein is required for replication ofthe virus.
  • Arrows indicate positions ofthe subgenomic promoters. Transcription ofthe selectable marker is under control ofthe TMN MP subgenomic promoter. Transcription ofthe inserted polynucleotide of interest is under control ofthe TMN CP subgenomic promoter.
  • the 3' portion ofthe construct includes TMV coat protein sequences and untranslated regions. These portions are optional.
  • Figure 5 presents a schematic diagram ofthe engineering of A1MN based viral constructs containing a polynucleotide of interest either as an independent open reading frame or as a genetic fusion with A1MN CP coding sequences.
  • the upper portion ofthe figure shows the genomic organization of R ⁇ A3 of A1MN, which includes genes encoding CP and MP as well as containing 5' and 3' UTRs and a subgenomic promoter.
  • the left side ofthe figure shows a construct in which transcription of an mR ⁇ A containing separate open reading frames that encode a polypeptide of interest (indicated as "target") and the A1MN CP is under control ofthe A1MN subgenomic promoter.
  • the right side ofthe figure shows a construct in which transcription of an mR ⁇ A containing a single open reading frame containing a polynucleotide of interest and CP coding sequences is under control ofthe A1MN CP subgenomic promoter.
  • the open reading frame encodes a fusion protein in which a polypeptide of interest is fused to CP.
  • Figures 6A-6E illustrate steps in a method for generating clonal root lines for expression of a polynucleotide of interest (indicated as "target” in the figure).
  • Figure 6E shows clonal root lines.
  • Figure 6F shows clonal root lines at a higher level of magnification.
  • Figure 6G shows GFP expression in a clonal root line, cells of which contain a viral vector that encodes GFP.
  • Figures 7 A - 7C show Western blot analyses demonstrating GFP production in 3 clonal root lines derived from plant cells into which a viral vector whose genome contains a gene that encodes GFP under control ofthe TMN CP promoter was introduced.
  • Figure 7 A shows GFP expression in the clonal root lines after 30 days of propagation in culture (i.e., 30 days after separation ofthe root from the leaf from which it was derived).
  • Figure 7B shows GFP expression in the clonal root lines after 60 days of propagation in culture (i.e., 60 days after separation ofthe root from the leaf from which it was derived).
  • C- represents control lanes containing no protein.
  • MWM represents molecular weight markers.
  • GFP-R represents samples from clonal root lines.
  • GFP-P represents GFP isolated from leaf tissue of a plant infected with the same construct used for generation ofthe clonal root lines.
  • Figure 7C is a control showing that the anti-GFP antibodies recognize commercially available GFP protein.
  • Figures 8A and 8B show photographs of clonal root lines producing hGH and GFP.
  • Figure 8 A shows a photograph of two clonal root lines taken under normal light conditions. The plate on the left shows a clonal root line derived from a plant cell into which a viral vector whose genome contains a gene that encodes human growth hormone (hGH) under control ofthe TMN CP promoter was introduced.
  • hGH human growth hormone
  • the plate on the right shows a clonal root line derived from a plant cell into which a viral vector whose genome contains a gene that encodes green fluorescent protein (GFP) under control ofthe TMN CP promoter was introduced.
  • Figure 8B shows a photograph ofthe same clonal root lines as shown in Figure 8 A taken under UN light, demonstrating expression of GFP.
  • GFP green fluorescent protein
  • Figure 9 shows a Western blot analysis to screen clonal root lines each derived from individual plant cells that were infected with a viral vector whose genome contains a gene that encodes human growth hormone (hGH) under control ofthe TMN CP promoter. Root lines were screened 30 days after separation ofthe root from the leaf from which it was derived. Root lines demonstrating high levels of expression are indicated with arrows. C- represents control lanes containing no protein. MWM represents molecular weight markers. hGH represents recombinant human growth hormone.
  • hGH human growth hormone
  • Figure 10 shows a Western blot analysis demonstrating hGH production in selected clonal root lines derived from plant cells into which a viral vector whose genome contains a gene that encodes hGH under control ofthe TMV CP promoter was introduced. The analysis was performed following 10 subculturmgs after separation of the roots from the leaves from which they were derived.
  • C- represents a control lane containing no protein.
  • MWM represents molecular weight markers.
  • hGH represents recombinant human growth hormone.
  • Figures IIA and IIB show Western blot analysis to screen clonal root lines each derived from individual plant cells that were infected with a viral vector whose genome contains a gene that encodes human growth hormone (GCSF) under control of the TMN CP promoter. Root lines were screened 30 days after separation ofthe root from the leaf from which it was derived. Root lines demonstrating high levels of expression are indicated with arrows. C- represents control lanes containing no protein. MWM represents molecular weight markers. GCSF represents recombinant human granulocyte colony stimulating factor.
  • GCSF human growth hormone
  • Figure 12 shows a Western blot analysis demonstrating GCSF production in selected clonal root lines derived from plant cells into which a viral vector whose genome contains a gene that encodes GCSF under control ofthe TMN CP promoter was introduced. The analysis was performed following 10 subculturmgs after separation ofthe roots from the leaves from which they were derived.
  • C- represents a control lane containing no protein.
  • MWM represents molecular weight markers.
  • GCSF represents recombinant human granulocyte colony stimulating factor.
  • Figure 13 illustrates steps in a method for generating clonal plant cell lines for expression of a polynucleotide of interest and identifying cell lines that display expression.
  • Figure 13 A shows a viral vector in which a polynucleotide of interest is inserted under control ofthe TMN CP promoter.
  • Figure 13B shows a protoplast suspension into which the vector was introduced (left plate) or a control protoplast suspension into which a vector lacking a GFP-encoding polynucleotide was introduced (right plate). The picture was taken under UN light and shows expression of GFP in the protoplasts containing the GFP-encoding expression vector.
  • Figure 13C shows a protoplast suspension into which the GFP-encoding vector was introduced. The photo was taken under UN light.
  • the inset ( Figure 13D) shows a higher magnification of GFP-expressing cells, also taken under UV light.
  • Figure 13E is a photograph showing enrichment for plant cell lines that express GFP. The photo was taken under normal light, of individual plant cell lines derived from the protoplast suspension shown in Figure 13C.
  • Figurel3F is a photograph ofthe same plates as shown in Figure 13C, taken under UN light. Cultures enriched for cell lines expressing GFP are evident as green fluorescing spots.
  • Figure 14 shows Western blot analyses demonstrating GCSF production in a clonal plant cell line derived from a plant cell into which a viral vector whose genome contains a gene that encodes GCSF under control ofthe TMV CP promoter was introduced.
  • Figure 14A shows a Western blot performed 48 hours after introduction of the vector.
  • Figure 14B shows a Western blot performed using the same cell lines as shown in Figure 14A performed 57 days after inoculation.
  • GCSF-COM indicates a lane in which recombinant GCSF protein was loaded as a positive control.
  • MWM indicates molecular weight markers.
  • C- indicates a lane in which plant extract made from plants not expressing GCSF was loaded.
  • Figure 15 shows GFP production in plant cell lines derived from plant cells into which a viral vector whose genome contains a gene that encodes GFP under control ofthe TMV CP promoter was introduced.
  • Figure 15 A shows enrichment for plant cell lines that express GFP.
  • Figure 15B shows a callus derived from a clonal plant cell line into which a similar viral vector, not encoding GFP, was introduced. The photographs were taken 3 months after the vector was introduced into the cells from which the clones in Figure 15A were derived. Both photographs were taken under UN light.
  • Figure 16 A shows a clonal plant that was obtained from a clonal root line derived from a plant cell into which a viral vector encoding hGH was introduced.
  • Figure 16B shows lesion formation in a sensitive host plant that was inoculated with a small leaf sample from the clonal plant, indicating that the clonal plant regenerated from the clonal root line maintains active viral replication.
  • a small leaf sample was used to inoculate a tobacco variety that is a host for formation of local lesions. Formation of lesions within 2 days of inoculation (see arrows) indicates that the clonal plant line regenerated from a clonal root line maintains active virus replication.
  • Figure 17 presents a schematic representation of certain families of viruses that infect plants.
  • Figure 18 shows representative examples of tobamo virus genomes.
  • Approximately in reference to a number includes numbers that fall within a range of 5% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Where ranges are stated, the endpoints are included within the range unless otherwise stated or otherwise evident from the context. [0031] Clonal: For the purpose ofthe present invention, the term clonal as applied, e.g., to a plant or plant tissue such as a root, leaf, stem, etc., means that the plant or plant tissue was derived from a single ancestral cell.
  • the cells or a clonal plant or plant tissue will be genetically identical with the exception of somatic mutations or other genetic alterations that may arise in descendant cells (e.g., through either natural or artificial introduction of a new gene into a descendant cell, telomere shortening, etc.).
  • the genome ofthe cells will be at least 95% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.9% identical.
  • Gene For the purposes ofthe present invention, the term gene has its meaning as understood in the art. In general, a gene is taken to include gene regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences, in addition to coding sequences (open reading frames). It will further be appreciated that the definition of gene can include nucleic acids that do not encode proteins but rather provide templates for transcription of functional RNA molecules such as tRNAs, rRNAs, microRNAs (miRNAs), short hairpin RNAs (shRNAs), short interfering RNAs, (siRNAs), etc.
  • Gene product or expression product A gene product or expression product is, in general, an RNA transcribed from a gene or polynucleotide, or a polypeptide encoded by an RNA transcribed from the gene or polynucleotide.
  • Isolated refers to a compound or entity that is 1) separated from at least some ofthe components with which it is normally associated (e.g., purified); 2) synthesized in vitro; and/or 3) produced or prepared by a process that involves the hand of man.
  • operably linlced refers to a relationship between two nucleic acids or two polypeptides wherein the expression of one ofthe nucleic acids or polypeptides is controlled by, regulated by, modulated by, etc., the other nucleic acid or polypeptide.
  • the transcription of a nucleic acid sequence is directed by an operably linlced promoter sequence; post-transcriptional processing of a nucleic acid is directed by an operably linlced processing sequence; the translation of a nucleic acid sequence is directed by an operably linked translational regulatory sequence; the transport or localization of a nucleic acid or polypeptide is directed by an operably linlced transport or localization sequence; and the post- translational processing of a polypeptide is directed by an operably linlced processing sequence.
  • nucleic acid or polypeptide sequence that is operably linlced to a second nucleic acid or polypeptide sequence is covalently linlced, either directly or indirectly, to such a sequence, although any effective three-dimensional association is acceptable.
  • a single nucleic acid or polypeptide sequence can be operably linlced to multiple other sequences.
  • a single promoter can direct transcription of multiple RNA species.
  • Percent (%) identity In reference to polynucleotides, “percent (%) identity” is defined as the percentage of nucleotide residues in a polynucleotide sequence that are identical with the nucleotide residues in the specific nucleic acid sequence with which comparison is being made, after aligning the sequences and introducing gaps, as needed, to achieve the maximum percent sequence identity. In reference to polypeptides, “percent (%) identity” is defined as the percentage of amino acid residues in a polypeptide sequence that are identical with the amino acid residues in the specific polypeptide sequence with which comparison is being made, after aligning the sequences and introducing gaps, as needed, to achieve the maximum percent sequence identity.
  • Alignment can be performed in various ways known to those of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST- 2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length ofthe sequences being compared. US Publication No. 20030211568 describes a number of suitable methods.
  • Polynucleotide of interest As used herein, the term "polynucleotide of interest” refers to any target nucleic acid sequence to be expressed in plant cells, as described herein.
  • the polynucleotide of interest will be a protein-coding polynucleotide (in which case the encoded polypeptide may be referred to as a polypeptide or protein of interest) but may also be a sequence that provides a template for transcription of a structural RNA or an active RNA such as a ribozyme, interfering RNA strand, etc.
  • the polynucleotide will be a gene that is not expressed in nature in the relevant type of plant cell, or is not expressed at the level mat the polynucleotide is expressed when expression is achieved by intervention ofthe hand of man, as described herein.
  • the polynucleotide comprises gene sequences that are not naturally found in the relevant plant cell at all; often including gene sequences that are naturally found in other cell types or organisms.
  • a polynucleotide of interest is one that is not naturally associated with the vector sequences with which it is associated according to the present invention.
  • the word polynucleotide is used interchangeably with “nucleic acid” or “nucleic acid molecule” herein.
  • purified means separated from one or more compounds or entities, e.g., one or more compounds or entities with which it is naturally found.
  • a compound or entity may be partially purified, substantially purified, or pure, where it is pure when it is removed from substantially all other compounds or entities, i.e., is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.
  • a preparation may be considered substantially pure if the nucleic acid represents at least 50% of all nucleic acid molecules in the preparation, preferably at least 75%, yet more preferably at least 90%, or greater, as listed above, on a molecule per molecule basis, a w/w basis, or both.
  • a preparation may be considered substantially pure if the polypeptide represents at least 50% of all polypeptides in the preparation, preferably at least 75%, yet more preferably at least 90%, or greater, as listed above, on a molecule per molecule basis, a w/w basis, or both.
  • a partially or substantially purified nucleic acid or polypeptide may be removed from at least 50%, at least 60%, at least 70%, or at least 80%, at least 90%, etc., ofthe material with which it is naturally found, e.g., cellular material such as otlier cellular proteins and/or nucleic acids.
  • Recombinant A "recombinant" molecule refers to a molecule that has been altered by the hand of man or that is derived from (e.g., copied from) such a molecule.
  • a recombinant polynucleotide typically contains sequences that are not found joined together in nature and/or that differ from a naturally occurring sequence.
  • An amplified or assembled recombinant polynucleotide may be included in a suitable vector, and the vector can be used to transform a suitable cell, which may be referred to as a "recombinant cell”. The nucleotide may then be expressed in the recombinant cell to produce, e.g., a "recombinant polypeptide".
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • a recombinant nucleic acid e.g., a recombinant viral nucleic acid may be a viral nucleic acid in which one or more sequences present in the naturally occurring form has been deleted or replaced by a different sequence or into which a non-native sequence has been inserted,
  • a "recombinant polypeptide" typically contains sequences that are not found joined together in nature and/or that differ from a naturally occurring sequence.
  • a recombinant polypeptide is a fusion protein, e.g., a protein containing two or more different proteins or peptides (which may be natural or synthetic and may be portions of a naturally occurring or synthetic polypeptide).
  • a recombinant polynucleotide that encodes a fusion protein may be created by removing the stop codon from the polynucleotide that encodes the first protein or peptide and appending a polynucleotide that encodes the second protein or peptide in frame, so that the resulting recombinant polynucleotide encodes a single recombinant polypeptide comprising the two proteins or peptides.
  • regulatory element or "regulatory sequence” in reference to a nucleic acid is generally used herein to describe a portion of nucleic acid that directs or increases one or more steps in the expression (particularly transcription, but in some cases other events such as splicing or other processing) of nucleic acid sequence(s) with which it is operatively linlced.
  • the term includes promoters and can also refer to enhancers and other transcriptional control elements. Promoters are regions of nucleic acid that include a site to which RNA polymerase binds before initiating transcription and that are typically necessary for even basal levels of transcription to occur. Generally such elements comprise a TATA box.
  • Enhancers are regions of nucleic acid that encompass binding sites for protein(s) that elevate transcriptional activity of a nearby or distantly located promoter, typically above some basal level of expression that would exist in the absence ofthe enhancer.
  • regulatory sequences may direct constitutive expression of a nucleotide sequence (e.g., expression in most or all cell types under typical physiological conditions in culture or in an organism); in other embodiments, regulatory sequences may direct cell or tissue- specific and/or inducible expression. For example, expression may be induced by the presence or addition of an inducing agent such as a hormone or other small molecule, by an increase in temperature, etc. Regulatory elements may also inhibit or decrease expression of an operatively linlced nucleic acid.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein. Such methods include Northern blotting, in situ hybridization, RT-PCR, sequencing, immunological methods such as immunoblotting, immunodetection, or fluorescence detection following staining with fluorescently labeled antibodies, oligonucleotide or cDNA microarray or membrane array, protein array analysis, mass spectrometry, etc.
  • a convenient way to determine expression level is to place a nucleic acid that encodes a readily detectable marker (e.g., a fluorescent or luminescent protein such as green fluorescent protein or luciferase, an enzyme such as alkaline phosphatase, etc.) in operable association with the regulatory element in an expression vector, introduce the vector into a cell type of interest or into an organism, maintain the cell or organism for a period of time, and then measure expression ofthe readily detectable marker, talcing advantage of whatever property renders it readily detectable (e.g., fluorescence, luminescence, alteration of optical property of a substrate, etc.). Comparing expression in the absence and presence ofthe regulatory element indicates the degree to which the regulatory element affects expression of an operatively linked sequence.
  • a readily detectable marker e.g., a fluorescent or luminescent protein such as green fluorescent protein or luciferase, an enzyme such as alkaline phosphatase, etc.
  • Self-replicate refers to the ability of a vector to copy itself inside a host cell.
  • a vector that can “self-replicate” carries sufficient information in its own genetic elements that it does not rely on other genetic elements (e.g., those utilized by the host cell to replicate its own genome) for its replication.
  • a vector that can self-replicate is one that includes at least one replicase gene such as an RNA polymerase and possibly additional replicase genes such as a helicase, methyltransferase, etc.
  • additional sequences, present in cis i.e., as part ofthe vector sequence) are required or can facilitate self-replication.
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cos id or viral vector.
  • the vector may be capable of autonomous replication.
  • a vector may provide one or more components necessary or sufficient for self-replication, or for replication or integration of another piece of nucleic acid.
  • Vectors are typically nucleic acids, and may comprise DNA and/or RNA. Preferred vectors are maintained extrachromosomally.
  • Viral nucleic acid refers to the genome of a virus, or a portion thereof (or, in the case of viruses whose genome comprises multiple segments, any ofthe segments or a portion of such a segment). The term encompasses both RNA and DNA forms of such nucleic acids and molecules having complementary sequences. DNA molecules identical to or complementary to viral RNA nucleic acids are considered viral nucleic acids, and RNA molecules identical to or complementary to viral DNA nucleic acids are considered viral nucleic acids, it being understood that DNA and RNA will contain T and U, respectively, at corresponding positions.
  • a viral nucleic acid may include one or more portions of non- viral origin (e.g., part or all of a naturally occurring gene, an entirely artificial sequence, or a combination of naturally occurring and artificial sequences) and may include portion(s) from multiple different virus types.
  • Viral replicon refers to a nucleic acid molecule comprising a portion or portions (cis sequences) sufficient for replication ofthe nucleic acid by viral replicase genes. Typically such sequences include a recognition site for a viral polymerase, e.g., a viral RNA polymerase in the case of viral replicons based on RNA viruses.
  • the present invention provides systems for expressing a polynucleotide or polynucleotides of interest in clonal root lines, clonal root cell lines, clonal plant cell lines (e.g., cell lines derived from leaf, stem, etc.), and in clonal plants.
  • the polynucleotide of interest is introduced into an ancestral plant cell using a plant viral vector whose genome includes the polynucleotide of interest operably linlced to (i.e., under control of) a promoter.
  • a clonal root line or clonal plant cell line is established from the cell containing the virus according to any of several techniques further described below.
  • the plant virus vector or portions thereof can be introduced into the plant cell by infection, by inoculation with a viral transcript or infectious cDNA clone, by electroporation, by T-DNA mediated gene transfer, etc.
  • the following sections describe plant viruses, plant viral vector, and methods for creating plant viral vectors for use in the present invention.
  • the inventive methods for generating clonal root lines, clonal root cell lines, clonal plant cell lines, and clonal plants that express a polynucleotide of interest are men described.
  • a “root line” is distinguished from a “root cell line” in that a root line produces actual rootlike structures or roots while a root cell line consists of root cells that do not form rootlike structures.
  • the use ofthe term “line” is intended to indicate that cells ofthe line can proliferate and pass genetic information on to progeny cells. Cells of a cell line typically proliferate in culture without being part of an organized structure such as those found in an intact plant.
  • the use ofthe term “root line” is intended to indicate that cells in the root structure can proliferate without being part of a complete plant. It is noted that the term “plant cell” encompasses root cells.
  • plant cell and “plant cell line” as used herein generally refer to cells and cell lines that consist of non-root plant tissue.
  • the plant cells can be, for example, leaf, stem, shoot, flower part, etc.
  • seeds can be derived from the clonal plants generated as derived herein. Such seeds will also contain the viral vector as will plants obtained from such seeds. Methods for obtaining seed stocks are well known in the art. See, e.g., U.S.S.N. 10/294,314.
  • FIG. 17 presents a schematic representation of certain families of viruses that infect plants.
  • Appendix A provides a representative list of plant virus families, based on the type of nucleic acid (e.g., dsDNA, ssDNA, ssRNA, dsRNA, or unassigned) that makes up the viral genome. Additional information can be found, for example, in The Classification and Nomenclature of Viruses", Sixth Report ofthe International Committee on Taxonomy of Viruses" (Ed.
  • plant viruses need to cross the cell wall, in addition to protective layers of waxes and pectins. Most or all plant viruses are thought to rely on mechanical breach ofthe cell wall, rather than on cell- wall-surface receptors, to enter a cell. Such a breach can be caused, for example, by physical damage to the cell, by an organism such as a bacterium, a fungus, a nematode, an insect, or a mite that can deliver the virus. In the laboratory, viruses are typically administered to plant cells simply by rubbing the virus on the plant. [0055] Some plant viruses have segmented genomes, in which two or more physically separate pieces of nucleic acid together make up the plant genome.
  • RNA plant virus genomes can be classified as mono-, bi-, or tri-partite, i.e., they may consist of 1, 2, or 3 nucleic acids respectively. In some cases, these separate pieces are packaged together in the same viral capsid; in others (i.e., those with multipartite genomes), each genome segment is packaged into its own viral particle. Infection can typically be accomplished by delivery either of plant viral nucleic acid (e.g., RNA) or capsid containing such nucleic acid.
  • plant viral nucleic acid e.g., RNA
  • the virus Once the virus has entered (infected) a cell, it typically replicates within the infected cell and then spreads locally (i.e., from cell to cell within leaves that were infected initially). Following local spread, the virus may move into uninfected leaves, e.g., upper leaves ofthe plant, which is referred to as systemic infection or systemic spread.
  • systemic infection or systemic spread In general, cell-to-cell spread of many plant viruses requires a functional movement protein (which allows movement of viral transcripts) while systemic spread requires a functional coat protein (and, generally, also a functional movement protein), which allows the formation of viral particles.
  • the viral genome may contain additional components that are required for local (e.g., cell-to-cell) or long distance (e.g., systemic) spread or facilitate such spread.
  • These exacting components may be either coding or noncoding components.
  • they may correspond to portions of a 3' untranslated region (UTR, also referred to as NTR) of a viral transcript (i.e., they may provide a template for transcription of a 3' untranslated region of a viral transcript).
  • UTR 3' untranslated region
  • viral transcript i.e., they may provide a template for transcription of a 3' untranslated region of a viral transcript.
  • important viral components can be either coding or noncoding regions of a viral genome and include a variety of regulatory regions. Such regions may function in replication and/or processing or expression of mRNA.
  • functional protein encoding component is meant a polynucleotide comprising a coding portion that encodes a functionally active protein, operably linlced to sufficient
  • viruses In order to successfully establish either a local (intraleaf) or systemic infection a virus must be able to replicate.
  • Many viruses contain genes encoding one or more proteins that participate in the replication process (referred to herem as replication proteins or replicase proteins).
  • replication proteins or replicase proteins proteins that participate in the replication process
  • RNA plant viruses encode an RNA polymerase. Additional proteins may also be required, e.g., helicase or methyltransferase protein(s).
  • the viral genome or segment may contain various sequence components, e.g., m-acting sequences, in addition to functional genes encoding replication proteins, which are also required for or facilitate replication.
  • Viral genomes or segments may also contain cw-acting sequences that contribute to high levels of transcript and/or expression.
  • nucleic acids encoding various viral proteins may be present within different viral nucleic acid molecules, which may complement each other in trans.
  • viral proteins e.g., replicase proteins, movement protein, coat protein
  • nucleic acid molecules may complement each other in trans.
  • WO 00/25574 and co-pending U.S. National Application Ser. No. 10/770,600, entitled “SYSTEM FOR EXPRESSION OF GENES IN PLANTS", filed February 3, 2004.
  • multiple different vectors are delivered which, together, allow for replication (and, optionally cell-to-cell and/or long distance movement) ofthe viral vector(s).
  • Some or all ofthe proteins may be encoded by the genome of transgenic plants.
  • Viral vectors based on any virus that infects plants may be used to generate a clonal root line, clonal plant cell line or clonal plant that expresses a polynucleotide of interest in accordance with the present invention.
  • Particularly preferred viruses are ssRNA viruses, most desirably with a (+)-stranded genome. Techniques and reagents for manipulating the genetic material present in such viruses are well known in the art.
  • a DNA copy ofthe viral genome is prepared and cloned into a microbial vector, particularly a bacterial vector.
  • Certain ssDNA viruses, including particularly geminiviruses may also be used.
  • viral vectors and viral nucleic acids such as viral genomes may exist in RNA or DNA form.
  • a feature such as a genome or portion thereof of an RNA virus, which is present within a DNA vector
  • the feature is present as the DNA copy ofthe RNA form.
  • Preferred vectors are based on viruses such as members ofthe Bromoviridae (e.g., bromoviruses, alfamoviruses, ilarviruses) and Tobamoviridae.
  • Certain preferred virus species include, for example, Alfalfa Mosaic Virus (AIMV), Apple Chlorotic Leaf Spot Virus, Apple Stem Grooving Virus, Barley Stripe Mosiac Virus, Barley Yellow Dwarf Virus, Beet Yellow Virus, Broad Bean Mottle Virus, Broad Bean Wilt Virus, Brome Mosaic Virus.
  • AIMV Alfalfa Mosaic Virus
  • Apple Chlorotic Leaf Spot Virus Apple Stem Grooving Virus
  • Barley Stripe Mosiac Virus Barley Yellow Dwarf Virus
  • Beet Yellow Virus Broad Bean Mottle Virus
  • Broad Bean Wilt Virus Brome Mosaic Virus.
  • MN Carnation Latent Virus
  • Carnation Mottle Virus Carnation Ringspot Virus
  • Carrot Mottle Virus Cassava Latent Virus
  • CLV Cowpea Chlorotic Mottle Virus
  • Cowpea Mosaic Virus CPMV
  • Cucumber Green Mottle Mosaic Virus Cucumber Mosaic Virus
  • Lettuce Infectious Yellow Virus Maize Chlorotic Mottle Virus
  • Maize Rayado Fino Virus Maize Streak Virus
  • MSV Maize Streak Virus
  • Parsnip Yellow Fleck Virus Pea Enation Mosaic Virus
  • Potato Virus X Potato Virus Y
  • Raspberry Bushy Dwarf Virus Rice Necrosis Virus
  • RMV Rice Stripe Virus
  • Rice Tungro Spherical Virus Ryegrass Mosaic Virus
  • Soil-borne Wheat Mosaic Virus Southern Bean Mosaic Virus
  • Tobacco Etch Virus TMV
  • TMV Tobacco Mosa
  • TMV is the type member of the tobamo virus group.
  • Tobamoviruses have single-(+)-stranded RNA genomes, and produce rod-shaped virions consisting ofthe RNA genome and coat protein (CP) polypeptides.
  • Tobamovirus genomes encode 4-5 polypeptides. Two ofthe polypeptides are translated from the same 5 '-proximal initiation codon and function in viral replication. These polypeptides include an RNA-dependent RNA polymerase,.
  • polypeptides having methyltransferase and RNA helicase activity are typically encoded.
  • the other encoded proteins typically include a movement protein and the coat protein, each of which is translated from a separate subgenomic RNA.
  • Representative examples of tobamovirus genomes are depicted in Figure 18.
  • Tobamoviruses otlier than TMV can be used in various embodiments ofthe invention.
  • the TMV genome is 6395 nucleotides long and is encapsidated with a 17.5 kD CP, which produces 300 nm-long rods.
  • TMV has three nonstructural proteins: 183 and 126 IcD proteins are translated from genomic RNA and are required for viral replication.
  • the 30 IcD movement protein provides for the transfer of viral RNA from cell-to-cell.
  • Plant species susceptible to infection with TMV include Beta vulgar is, Capsicum frutescens, Chenopodium amaranticolor, Chenopodium hybridum, Chenopodium quinoa, Cucumis melo, Cucumis sativus, Cucurbita pepo, Datura stramonium, Lactuca sativa, Lucopersicon esculentum, Lycopersicon pimpinellifolium, Nicotiana benthamiana, Nicotiana bigelovii, Nicotiana clevelandii, Nicotiana debneyi, Nicotiana glutinosa, Nicotiana rustica, Nicotiana sylvestris, Nicotiana tabacum, Papaver nudicaule, Phaseolus vulgaris, Physalis floridana, Physalis peruviana, andSolanum tuber osum.
  • an AlMV-based viral vector (viral nucleic acid) is used.
  • AIMV is mAlfamovirus, closely related to the Ilarvirus group and is a member ofthe Bromoviridae family.
  • the genome of AIMV consists of three positive-sense RNAs (RNAs 1-3).
  • RNAs 1 and 2 encode replicase proteins PI and P2, respectively;
  • RNA3 encodes the cell-to-cell movement protein P3.
  • a subgenomic RNA, RNA4, is synthesized from RNA3. This subgenomic RNA4 encodes the viral coat protein (CP).
  • AIMV depends on a functional P3 protein for cell-to-cell movement, and requires the CP protein throughout infection.
  • virions of AIMV can vary significantly in size (e.g., 30- to 60-nm in length and 18 nm in diameter) and form (e.g., spherical, ellipsoidal, or bacilliform).
  • AIMV The host range of AIMV is remarkably wide and includes the agriculturally valuable crops alfalfa (Medicago sativa), tomato (Lycopersicon esculentum), lettuce (Lactuca sativa), common bean (Phaseolus vulgaris), potato (Solarium tuberosum), white clover (Trifolium repens) and soybean (Glycine max).
  • Particular susceptible host species include, for example, Abelmoschus esculentus, Ageratum conyzoides, Amaranthus caudatus, Amaranthus retrofiexus, Antirrhinum majus, Apium graveolens, Apium graveolens var.
  • rapaceum Arachis hypogaea, Astragalus glycyphyllos, Beta vulgaris, Brassica campestris ssp. rapa, Calendula officinalis, Capsicum annuum, Capsicum frutescens, Caryopteris incana, Catharanthus roseus, Celosia argentea, Cheiranthus cheiri, Chenopodium album, Chenopodium amaranticolor, Chenopodium murale, Chenopodium quinoa, Cicer arietinum, Cichorium endiva, Coriandrum sativum, Crotalaria spectabilis, Cucumis melo, Cucumis sativus, Cucurbita pepo, Cyamopsis tetragonoloba, Daucus carota (var.
  • Zinnia elegans Zinnia elegans. While AIMV is a preferred viral vector, other alfamoviruses can also be used in various embodiments ofthe invention. Related viruses, such as ilarviruses can also be used.
  • a viral vector is a viral nucleic acid.
  • the viral vector is the genome, or a majority thereof (i.e., at least 50% ofthe genome), of a virus, or a nucleic acid molecule complementary in base sequence to such a nucleic acid molecule.
  • the viral vector may be a genome segment, or a majority thereof.
  • the viral vector may be in RNA or DNA form.
  • the viral vector comprises a portion sufficient to support replication ofthe viral vector in the presence ofthe appropriate viral replicase proteins, i.e., constitutes a viral replicon.
  • the ability of any particular portion of a viral genome to support replication of a nucleic acid that includes the portion, in the presence of viral replicase proteins can readily be tested using methods known in the art, e.g., by making deletion mutants, by transferring the portion into a nucleic acid that does not support replication and determining whether replication occurs, etc.
  • the replicase proteins may be encoded by the vector, by another vector, or by a plant into which the vector is introduced.
  • the vector is capable of self-replication, i.e., it encodes the necessary viral proteins for replication of the virus within an appropriate plant host.
  • the vector comprises a MP gene.
  • the vector comprises a CP gene.
  • neither an MP gene nor a CP gene is present in the vector. Since the clonal root lines, clonal plant lines, and clonal plants are derived from single ancestral cells into which the vector has been introduced, it is not necessary for the viral vector to have cell-to-cell or long distance movement capability. In particular, clonal plants can express the polynucleotide of interest throughout the plant even though the viral transcript does not move, since each cell is derived from a single ancestral cell that contains the viral vector.
  • a polynucleotide of interest is inserted into a viral vector under control of (i.e., operably linlced to), a promoter that directs transcription ofthe polynucleotide in a plant cell of interest.
  • a plant viral promoter is used, e.g., a promoter for coat protein, movement protein, etc.
  • the polynucleotide of interest may be inserted in place ofthe endogenous MP or CP coding sequence.
  • a TMV-based vector in which the TMV CP coding sequence has been replaced by a polynucleotide of interest, under control ofthe TMV CP promoter can be used.
  • the inserted polynucleotide may include its own promoter, which may be identical or similar to one ofthe naturally occurring viral promoters, may be from a different virus (e.g., the cauliflower mosaic virus), may be a non-viral promoter such as a promoter for a plant gene, or a synthetic promoter.
  • an inducible promoter is used.
  • a variety of inducible promoters are known that function in plants. See, e.g., Zuo, J.
  • trans- activation is used to induce or increase expression of a polynucleotide of interest.
  • the expression cassette comprising the polynucleotide can be an inactive expression cassette that comprises an inactive or silenced foreign nucleic acid sequence, which is capable of directing expression of a polynucleotide of interest upon its activation.
  • trans-activation is accomplished by introducing a factor for activating or facilitating the expression of an inactive or silenced polynucleotide sequence into cells ofthe clonal entity.
  • a promoter that can be activated in trans in such a manner is referred to as being "trans-activatable". See U.S.S.N.
  • Such methods include techniques based on recombination (e.g., using a Lox/Cre or Flp/Frt recombinase system) and techniques based on proteins comprising a DNA binding domain such as GAL4 and a transcriptional activation domain such as VP16.
  • recombination e.g., using a Lox/Cre or Flp/Frt recombinase system
  • proteins comprising a DNA binding domain such as GAL4 and a transcriptional activation domain such as VP16.
  • GAL4 a DNA binding domain
  • VP16 transcriptional activation domain
  • the polynucleotide is inserted to create an independent open reading frame, while in other embodiments ofthe invention the polynucleotide is inserted to create an open reading frame in which a polynucleotide lacking a stop codon is inserted in frame with sequences encoding part or all of a viral protein such as CP, so that a fusion protein is produced upon translation. Multiple polynucleotides can be inserted.
  • the TMV vector retains part or all of its 3' UTR and/or all or part ofthe CP coding sequence.
  • the polynucleotide of interest or a viral vector into which the polynucleotide of interest is inserted comprises a portion encoding a targeting sequence, e.g., a sequence that targets an encoded polypeptide to a particular intracellular organelle or compartment.
  • a targeting sequence e.g., a sequence that targets an encoded polypeptide to a particular intracellular organelle or compartment.
  • the secreted polypeptide can then be harvested from culture medium or from interstitial fluid of a plant tissue.
  • Figures 1 - 5 show examples of engineering various plant virus vectors suitable for use in the present invention.
  • Figure 1 shows a TMV based virus construct, D4, and the same construct following insertion of a polynucleotide of interest (e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target") whose transcription is under control ofthe TMV CP subgenomic promoter. Details regarding the creation of such vectors are given in Example 1.
  • a polynucleotide of interest e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target”
  • FIG. 2 presents a schematic diagram ofthe engineering of a TMV based viral construct containing a polynucleotide of interest.
  • the upper portion ofthe figure shows the genomic organization of a TMV based virus construct, 30B (Yusibov, V., Shivprasad, S., Turpen, T.H., Dawson, W., and Koprowski, H., "Plant viral vectors based on tobamoviruses", in Plant Biotechnology: New Products and Applications (Eds. J. Hammond, P. McGarvey, and N. Yusibov), pp.81-94, Springer-Nerlag, 1999).
  • the lower portion shows the same construct following insertion of a polynucleotide of interest (e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target”).
  • a polynucleotide of interest e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target”
  • the 126/183 kDa protein is required for replication ofthe virus.
  • the 30 IcD protein is the movement protein (MP) that mediates cell-to-cell movement.
  • CP is the coat protein that mediates systemic spread. Arrows indicate positions ofthe subgenomic promoters in certain embodiments ofthe invention. Transcription ofthe inserted polynucleotide is under control of an introduced promoter. CP expression is under control ofthe endogenous CP promoter in the construct shown in Figure 2.
  • polynucleotide of interest in frame with the CP coding sequence so as to encode a fusion protein can also be used.
  • polynucleotides of interest and their encoded proteins can be expressed as independent open reading frames (see, e.g., Pogue, G.P., Lindbo. J.A., Dawson, W.O., and Turpen, T.H. "Tobamovirus transient expression vectors: tools for plant biology and high-level expression of foreign proteins in plants", PI, Mol. Biol.
  • transcription of a polynucleotide of interest and/or an endogenous gene such as MP or CP can be driven by endogenous promoters or inserted promoters (which may be identical to naturally occurring vectors from the same or a different virus or may be synthetic, or a combination of natural and synthetic sequences.
  • endogenous promoters or inserted promoters which may be identical to naturally occurring vectors from the same or a different virus or may be synthetic, or a combination of natural and synthetic sequences.
  • the 3 ' portion of the construct preferably includes the TMV 3 ' UTR, which may form stem-loop structure(s) as shown.
  • the 3' portion ofthe construct may also include TMV coat protein sequences that contain a cis element that may be required for optimal replication. This sequence is optional.
  • Figure 3 presents a schematic diagram of the engineering of a TMV based viral construct containing a polynucleotide of interest and a gene encoding a marker, e.g., a marker that allows for detection and/or selection.
  • the upper portion ofthe figure shows the genomic organization of a TMV based virus construct, D4.
  • the middle portion ofthe figure shows the same construct after insertion of a gene encoding a detectable marker (GFP) replacing the MP coding sequence.
  • the lower portion ofthe figure shows the same construct following insertion of a polynucleotide of interest (e.g., a gene encoding hGH, GCSF, GFP, etc., indicated as "target").
  • the 126/183 IcDa protein is required for replication ofthe virus. Arrows indicate positions ofthe subgenomic promoters. Transcription ofthe detectable marker is under control ofthe MP subgenomic promoter. Transcription ofthe inserted polynucleotide of interest is under control ofthe TMV CP subgenomic promoter. However, other promoters could be used as described above.
  • the 3' portion ofthe construct includes TMV coat protein sequences that contain a cis element that may be required for optimal replication and that may form stem-loop structure(s) as shown.
  • Figure 4 shows a vector similar to that shown in Figure 3 except that a selectable marker (a gene encoding a protein that confers resistance to kanamycin) is inserted instead of a gene encoding GFP.
  • a selectable marker a gene encoding a protein that confers resistance to kanamycin
  • Including a gene that encodes a detectable or selectable marker in addition to a polynucleotide of interest is useful in the identification of clonal root lines and clonal plant cell lines that contain the vector and/or for identifying those lines that exhibit high and/or stable levels of expression.
  • a suitable marker for use in the invention is a detectable marker or a selectable marker.
  • the term “marker” can refer either to a nucleotide sequence, e.g., a gene, that encodes a product (protein) that allows for detection or selection, or can be used to refer to the protein itself.
  • selectable marker is used herein as it is generally understood in the art and refers to a marker whose presence within a cell or organism confers a significant growth or survival advantage or disadvantage on the cell or organism under certain defined culture conditions (selective conditions).
  • the conditions may be the presence or absence of a particular compound or a particular environmental condition such as increased temperature, increased radiation, presence of a compound that is toxic in the absence ofthe marker, etc.
  • growth advantage is meant either enhanced viability (e.g., cells or organisms with the growth advantage have an increased life span, on average, relative to otherwise identical cells), increased rate of proliferation (also referred to herein as “growth rate”) relative to otherwise identical cells or organisms, or both.
  • growth rate increased rate of proliferation relative to otherwise identical cells or organisms, or both.
  • a population of cells having a growth advantage will exhibit fewer dead or nonviable cells and/or a greater rate of cell proliferation that a population of otherwise identical cells lacking the growth advantage.
  • Antibiotic resistance markers are a non-limiting example of a class of selectable marker that can be used to select cells that express the marker. In the presence of an appropriate concentration of antibiotic (selective conditions), such a marker confers a growth advantage on a cell that expresses the marker.
  • a selectable marker of this type that is commonly used in plant cells is the NPTII protein, which encodes a protein that provides resistance against the antibiotic kanamycin.
  • Additional selectable markers include proteins that confer resistance against carbenecillin (e.g., ⁇ -lactamases), proteins that confer resistance against gentamicin, hygronycin, etc.)
  • a second non-limiting class of selectable markers are nutritional markers.
  • Such markers are generally enzymes that function in a biosynthetic pathway to produce a compound that is needed for cell growth or survival. In general, under nonselective conditions the required compound is present in the environment or is produced by an alternative pathway in the cell. Under selective conditions, functioning ofthe biosynthetic pathway in which the marker is involved is needed to produce the compound.
  • a detectable marker is a marker whose presence within a cell can be detected through means other than subjecting the cell to a selective condition or directly measuring the level ofthe marker itself.
  • the expression of a detectable marker within a cell results in the production of a signal that can be detected and/or measured.
  • the process of detection or measurement may involve the use of additional reagents and may involve processing of the cell.
  • the detectable marker is an enzyme
  • detection or measurement ofthe marker will typically involve providing a substrate for the enzyme.
  • the signal is a readily detectable signal such as light, fluorescence, luminescence, bioluminescence, chemiluminescence, enzymatic reaction products, stainable products, or color.
  • detectable markers for use in the present invention include fluorescent proteins such as green fluorescent protein (GFP) and variants thereof.
  • suitable markers include luciferase, yellow fluorescent protein (YFP), lichenase, ⁇ - galactosidase, alkaline phosphatase, etc.
  • the detectable marker is one that can be detected in intact, living root and/or plant cells.
  • a preferred viral vector for use in the present invention is an AIMV vector in which a polynucleotide of interest is inserted, as shown in Figure 5.
  • the polynucleotide of interest may replace the native AIMV CP encoding component in RNA3 of AIMV. Transcription ofthe polynucleotide of interest may be placed under control ofthe AIMV CP promoter. Alternately, the polynucleotide may replace the AIMV MP encoding component, and its transcription may be placed under control ofthe AIMV MP promoter. In other embodiments the inserted polynucleotide does not replace endogenous viral sequences.
  • the polynucleotide of interest may be inserted in frame with CP coding sequences (complete or partial), so that a fusion protein is produced.
  • the fusion protein comprises a cleavage site between the CP portion and the remainder, so that the fusion protein can be cleaved to yield a protein of interest free of CP sequences (or containing only a small number of such sequences).
  • the fusion protein assembles into particles, which can facilitate purification and/or antigen presentation (see, e.g., U.S. Patent Nos. 6,042,832 and 6,448,070).
  • CMV cauliflower mosaic virus
  • a polynucleotide of interest is inserted under control ofthe CMV CP promoter, replacing the CMV CP encoding component found in the genome of naturally occurring CMV.
  • certain sequences may enhance replication or expression, etc.
  • Such sequences may comprise, for example, part or all of a viral transcript 5' or 3' UTR.
  • viral vectors will be prepared by altering an existing plant virus genome, for example by removing particular genes and/or by disrupting or substituting particular sequences so as to inactivate or replace them. In such circumstances, the vectors will show very high sequence identity with natural viral genomes.
  • completely novel vectors may also be prepared, for example, by separately isolating individual desired genetic elements and linking them together, optionally with the inclusion of additional elements. It is noted that when a plant virus vector is said to affirmatively express a particular protein or activity needed for viral replication, movement, or some other viral function, it is not necessary that the relevant gene be identical to the corresponding gene found in nature.
  • the protein may be used in accordance with the present invention. Very high sequence identity with the natural protein, however, is generally preferred. For instance, large deletions (e.g., greater than about 25 amino acids) should generally be avoided according to certain embodiments of the invention.
  • viral proteins expressed in accordance with the present invention will show at least 50%, preferably 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the corresponding natural viral protein. More particularly, the inventive viral protein should typically show 100%) identity with critical functional portions (typically of at least several amino acids, often of at least 10, 20, 30, 40, 50 or more amino acids) ofthe relevant natural viral protein.
  • amino acids have been classified and divided into groups according to (1) charge (positive, negative, or uncharged); (2) volume and polarity; (3) Grantham's physico-chemical distance; and combinations of these.
  • amino acids may be divided into the following 6 categories based on volume and polarity: special (C); neutral and small (A, G, P, S, T); polar and relatively small (N, D, Q, E), polar and relatively large (R, H, K), nonpolar and relatively small (I, L, M, V), and nonpolar and relatively large (F, W, Y).
  • C special
  • A, G, P, S, T neutral and small
  • N, D, Q, E polar and relatively small
  • R, H, K polar and relatively large
  • I, L, M, V nonpolar and relatively small
  • F, W, Y nonpolar and relatively large
  • a conservative amino acid substitution may be defined as one that replaces one amino acid with an amino acid in the same group.
  • a variety of functionally equivalent proteins can be derived by making one or more amino acid substitutions, e.g., conservative amino acid substitutions, in a given viral protein.
  • the present invention provides methods for generating a clonal root line in which a plant viral vector is used to direct expression of a polynucleotide of interest.
  • Figures 6A-6E show steps in the method according to certain embodiments ofthe invention.
  • one or more viral expression vector(s) including a polynucleotide of interest operably linlced to a promoter is introduced into a plant or a portion thereof according to any of a variety of known methods.
  • plant leaves can be inoculated with viral transcripts.
  • ssRNA may be prepared by transcription of a DNA copy ofthe genome, or by replication of an RNA copy, either in vivo or in vitro.
  • inventive ssRNA vectors will often be prepared by in vitro transcription, particularly with T7 or SP6 polymerase.
  • Infectious cDNA clones can also be used.
  • Agrobacterially mediated gene transfer can also be used to transfer viral nucleic acids such as viral vectors (either entire viral genomes or portions thereof) to plant cells using, e.g., agroinfiltration, according to methods known in the art.
  • the plant or plant portion is then maintained (e.g., cultured or grown) under conditions suitable for replication ofthe viral transcript.
  • the virus spreads beyond the initially inoculated cell, e.g., locally from cell to cell and/or systemically from an initially inoculated leaf into additional leaves.
  • the virus does not spread.
  • the viral vector may contain genes encoding functional MP and/or CP, but may be lacking one or both of such genes.
  • the viral vector is introduced into (infects) multiple cells in the plant or portion thereof.
  • Figure 6B shows a plant into which a viral vector (depicted schematically in Figure 6A) has been introduced.
  • leaves are harvested.
  • Figure 6C shows leaf portions after harvesting from a virus-infected plant.
  • leaves may be harvested at any time following introduction of the viral vector.
  • a clonal root culture (or multiple cultures) is prepared, e.g., by known methods further described below and in Example 2.
  • any available method may be used to prepare a clonal root culture from a plant or plant tissue into which a viral vector has been introduced.
  • Agrobacteria can transfer DNA to a large and diverse set of plant types including numerous dicot and monocot angiosperm species and gymnosperms (See, Gelvin, S.B., "Agrobacterium-Medi ⁇ Aed Plant Transformation: the Biology behind the "Gene- Jockeying" Tool", Microbiology and Molecular Biology Reviews, 67(1): 16-37 (2003) and references therein, all of which are incorporated herein by reference).
  • the molecular basis of genetic transformation of plant cells is transfer from the bacterium and integration into the plant nuclear genome of a region of a large tumor-inducing (Ti) or rhizogenic (Ri) plasmid that resides within various Agrobacterial species.
  • This region is referred to as the T-region when present in the plasmid and as T-DNA when excised from the plasmid.
  • T-region when present in the plasmid
  • T-DNA when excised from the plasmid.
  • a single-stranded T-DNA molecule is transferred to the plant cell in naturally occurring Agrobacterial infection and is ultimately incorporated (in double-stranded form) into the genome.
  • Systems based on Ti plasmids are widely used for introduction of foreign genetic material into plants and for production of transgenic plants.
  • A. tumefaciens causes crown gall disease while A. rhizogenes causes development of hairy roots at the site of infection, a condition known as "hairy root disease".
  • hairy root disease a condition known as "hairy root disease”.
  • Each root arises from a single genetically transformed cell.
  • root cells in the roots are clonal, and each root represents a clonal population of cells.
  • the roots produced by A. rhizogenes infection are characterized by a high growth rate and genetic stability. (Giri, A. and Narasu, M.L., Biotechnology Advances, 18: 1-22 (2000) and references therein, all of which are incorporated herein by reference).
  • the present invention encompasses the use of any strain of Agrobacteria, particularly A. rhizogenes strains, that is capable of inducing formation of roots from plant cells.
  • A. rhizogenes strains that is capable of inducing formation of roots from plant cells.
  • a portion ofthe Ri plasmid (Ri T-DNA) is responsible for causing hairy root disease. While transfer of this portion ofthe Ri plasmid to plant cells can conveniently be accomplished by infection with Agrobacteria harboring the Ri plasmid, the invention also encompasses the use of alternative methods of introducing the relevant region into a plant cell.
  • Such methods include any available method of introducing genetic material into plant cells including, but not limited to, biolistics, electroporation, PEG-mediated DNA uptake, Ti-based vectors, etc.
  • the relevant portions ofthe Ri T-DNA can also be introduced into plant cells by use of a viral vector.
  • the Ri genes can be included in the same vector that contains the polynucleotide of interest or in a different viral vector, which can be the same or a different type to that ofthe vector that contains the polynucleotide of interest. It is noted that the entire Ri T-DNA may not be required for production of hairy roots, and the invention encompasses the use of portions ofthe Ri T-DNA, provided that such portions contain sufficient genetic material to induce root formation, as known in the art.
  • Additional genetic material e.g., genes present within the Ri plasmid but not within the T-DNA, may also be transferred to the plant cell in accordance with the invention, particularly genes whose expression products facilitate integration ofthe T- DNA into the plant cell DNA.
  • the harvested leaf portions are contacted with A. rhizogenes under conditions suitable for infection and transformation.
  • Example 2 describes one method for generating root lines from leaves into which a viral vector has been introduced. The leaf portions are maintained in culture to allow development of hairy roots.
  • Figure 6D shows hairy roots generated by individual cells in leaf portions infected with A. rhizogenes.
  • Each root is clonal, i.e., cells in the root are derived from a single ancestral cell into which the Ri T-DNA was transferred.
  • a portion of such ancestral cells will also contain the viral vector.
  • cells in a root derived from such an ancestral cell will also contain the viral vector since it will be replicated and will be transmitted during cell division.
  • a high proportion, preferably at least 50%), more preferably at least 75%, at least 80%, at least 90%), at least 95%, or all (100%) or substantially all (at least 98%) ofthe cells will contain the viral vector. It is noted that since the viral vector is inherited by daughter cells within the clonal root, movement ofthe viral vector within the root is not necessary to maintain the viral vector throughout the root.
  • Individual clonal hairy roots may be removed from the leaf portion and further cultured, as shown in Figures 6E and 6F. Such roots are also referred to herein as root lines.
  • Figure 6E shows individual clonal roots placed in a line in a Petri dish.
  • Figure 6F shows the same root lines at higher magnification. The roots continue to grow. These roots were derived from plants into which a viral vector containing a GFP gene had been introduced.
  • Figure 6G shows a photograph of single root taken under UN light. Expression of GFP throughout the root is evident.
  • Root lines As described in Examples 2 - 4, a variety of different clonal root lines have been generated using the inventive methods. These root lines were generated using viral vectors containing polynucleotides of interest encoding GFP, hGH, and GCSF. The root lines were tested by Western blot. Root lines displayed a variety of different expression levels ofthe various polypeptides. Root lines displaying high expression were selected and further cultured. These root lines were subsequently tested again and shown to maintain high levels of expression over extended periods of time, indicating stability. The level of expression was comparable to or greater than expression in intact plants infected with the same viral vector used to generate the clonal root lines.
  • the stability of expression ofthe root lines was superior to that obtained in plants infected with the same viral vector.
  • the root lines may be cultured on a large scale for production of polypeptides of interest as discussed further below. It is noted that the clonal root lines (and cell lines derived from the clonal root lines) can generally be maintained in medium that does not include various compounds, e.g., plant growth hormones such as auxins, cytokinins, etc., that are typically employed in the culture of root and plant cells. This feature greatly reduces the expense associated with tissue culture, and the inventors expect that it will contribute significantly to the economic feasibility of protein production using plants.
  • plant growth hormones such as auxins, cytokinins, etc.
  • any of a variety of methods may be used to select clonal roots that express a polynucleotide of interest.
  • Western blots, ELISA assays, etc. can be used to detect an encoded polypeptide.
  • detectable markers such as GFP
  • alternative methods such as visual screens can be performed.
  • an appropriate selection can be imposed (e.g., the leaf material and/or roots derived therefrom can be cultured in the presence of an appropriate antibiotic or nutritional condition and surviving roots identified and isolated).
  • Certain viral vectors contain two or more polynucleotides of interest, e.g., two or more polynucleotides encoding different polypeptides. If one of these is a selectable or detectable marker, clonal roots that are selected or detected by selecting for or detecting expression ofthe marker will have a high probability of also expressing the second polynucleotide. Screening for root lines that contain particular polynucleotides can also be performed using PCR and other nucleic acid detection methods.
  • clonal root lines can also be screened for presence ofthe virus by inoculating host plants that will form local lesions as a result of virus infection (e.g., hypersensitive host plants). For example, 5 mg of root tissue can be homogenized in 50 ul of phosphate buffer and used to inoculate a single leaf of a tobacco plant. If the virus is present in root cultures, within two to tliree days characteristic lesions will appear on the infected leaves. This means that the root line contains recombinant virus that carries the polynucleotide of interest (target gene). If no local lesions are formed, there is no virus, and the root line is rejected as negative. This method is highly time and cost efficient.
  • roots that contain the virus are subjected to secondary screening, e.g., by Western blot or ELISA to select high expressers. Additional screens, e.g., screens for rapid growth, growth in particular media or under particular environmental conditions, etc., can also be applied. These screening methods may, in general, be applied in the development of any ofthe clonal root lines, clonal root cell lines, clonal plant cell lines, and/or clonal plants described herein. [00100] As will be evident to one of ordinary skill in the art, a variety of modifications may be made to the above description ofthe inventive methods for generating clonal root lines that contain a viral vector. Such modifications are within the scope ofthe invention.
  • the Ri-DNA is introduced prior to introducing the viral vector.
  • multiple different viral vectors are introduced.
  • Such vectors may, for example, trans-complement each other with respect to functions such as replication, cell-to-cell movement, and/or long distance movement.
  • the vectors may contain different polynucleotides of interest, e.g., polynucleotides that encode individual polypeptides that associate to form a single protein complex such as antibodies, etc., or polynucleotides that encode different enzymes in a biosynthetic pathway. Selection for roots that express multiple polypeptides of interest may be performed as described above for single polynucleotides or polypeptides. [001 3] D.
  • the invention provides methods for generating clonal root lines, wherein cells in the root lines contain a viral vector.
  • a variety of different cell lines can be generated from roots.
  • root cell lines can be generated from individual root cells obtained from the root using a variety of known methods. Such root cell lines may be obtained from various different root cell types within the root.
  • root material is harvested and dissociated (e.g., physically and/or enzymatically digested) to release individual root cells, which are then further cultured. Complete protoplast formation is generally not necessary.
  • root cells can be plated at very dilute cell concentrations, so as to obtain root cell lines from single root cells.
  • Root cell lines derived in this manner are clonal root cell lines contain the viral vector. Such root cell lines therefore exhibit stable expression ofthe polynucleotide of interest.
  • Clonal plant cell lines can also be obtained in a similar manner from the clonal roots, e.g., by culturing dissociated root cells in the presence ofthe appropriate plant hormones. Screens and successive rounds of enrichment can be used to identify cell lines that express the polynucleotide of interest at high levels. However, if the clonal root line from which the cell line is derived already expresses at high levels, such additional screens may be unnecessary.
  • cells of a clonal root cell line are derived from a single ancestral cell that contains the viral vector and will, therefore, also contain the viral vector since it will be replicated and will be transmitted during cell division.
  • a high proportion preferably at least 50%, more preferably at least 75%, at least 80%, at least 90%, at least 95%, or all (100%) or substantially all (at least 98%) ofthe cells will contain the viral vector.
  • the clonal root cell lines can be used for production of a polynucleotide of interest as described below.
  • the present invention provides methods for generating a clonal plant cell line in which a plant viral vector is used to direct expression of a polynucleotide of interest.
  • one or more viral expression vector(s) including a polynucleotide of interest operably linked to a promoter is introduced into cells of a plant cell line that is maintained in cell culture.
  • a number of plant cell lines from various plant types are known in the art, any of which can be used. Newly derived cell lines can also be generated according to Icnown methods for use in practicing the invention.
  • a viral vector is introduced into cells ofthe plant cell line according to any of a number of methods.
  • Figure 13 shows steps in a method for generating clonal plant cell lines in accordance with the invention.
  • Figure 13 A shows a viral vector suitable for introduction into plant cells (e.g., protoplasts).
  • the plant cell line may be maintained in tissue culture, e.g., as shown in Figures 13B and 13C.
  • the viral vector may replicate, and polynucleotides of interest may be expressed.
  • Clonal plant cell lines are derived from the culture, e.g., by a process of successive enrichment.
  • samples may be removed from the culture, optionally with dilution so that the concentration of cells is low, and plated in Petri dishes in individual droplets. The droplets are then maintained to allow cell division.
  • the droplets may contain a variable number of cells, depending on the initial density ofthe culture and the amount of dilution.
  • the cells can be diluted such that most droplets contain either 0 or 1 cell if it is desired to obtain clonal cell lines expressing the polynucleotide of interest after only a single round of enrichment.
  • it can be more efficient to select a concentration such that multiple cells are present in each droplet and then screen the droplets to identify those that contain expressing cells.
  • any appropriate screening procedure can be employed. For example, selection or detection of a detectable marker such as GFP can be used.
  • Figure 13F is a photograph taken under UN light and showing individual droplets in which cell lines expressing GFP from a viral vector are present.
  • a diversity of viral vectors containing one or more polynucleotides of interest can be used as can combinations of multiple different vectors. Similar screening methods can also be used.
  • cells of a clonal plant cell line are derived from a single ancestral cell that contains the viral vector and will, therefore, also contain the viral vector since it will be replicated and will be transmitted during cell division.
  • a high proportion preferably at least 50%, more preferably at least 75%, at least 80%, at least 90%, at least 95%, or all (100%o) or substantially all (at least 98%) ofthe cells will contain the viral vector.
  • the clonal plant cell line can be used for production of a polypeptide of interest as described below.
  • Clonal plants can be generated from the clonal roots, clonal root cell lines, and/or clonal plant cell lines produced according to the various methods described above. Methods for the generation of plants from roots, root cell lines, and plant cell lines such as the clonal root lines, clonal root cell lines, and clonal plant cell lines described herein are well known in the art (See, e.g., Peres et al., Plant Cell, Tissue, and Organ Culture 65, 37-44, 2001 and standard reference works on plant molecular biology and biotechnology cited elsewhere herein.
  • the invention therefore provides a method of generating a clonal plant comprising steps of (i) generating a clonal root line, clonal root cell line, or clonal plant cell line according to any ofthe inventive methods described above; and (ii) generating a whole plant from the clonal root line, clonal root cell line, or clonal plant.
  • the clonal plants may be propagated and grown according to standard methods.
  • Example 7 describes generation of a clonal plant from a clonal root line containing a viral vector that encodes human growth hormone.
  • the cells of a clonal plant are derived from a single ancestral cell that contains the viral vector and will, therefore, also contain the viral vector since it will be replicated and will be transmitted during cell division.
  • a high proportion preferably at least 50%, more preferably at least 75%>, at least 80%, at least 90%>, at least 95%, or all (100%) or substantially all (at least 98%) ofthe cells will contain the viral vector. It is noted that since the viral vector is inherited by daughter cells within the clonal plant, movement ofthe viral vector is not necessary to maintain the viral vector.
  • Any plant susceptible to viral infection may be utilized in accordance with the present invention.
  • plants that are amenable to growth under defined conditions for example in a greenhouse and/or in aqueous systems. It may also be desirable to select plants that are not typically consumed by human beings or domesticated animals and/or are not typically part ofthe human food chain, so that they may be grown outside without concern that the expressed polynucleotide may be undesirably ingested. In other embodiments, however, it will be desirable to employ edible plants.
  • certain desirable plant characteristics will be determined by the particular polynucleotide to be expressed.
  • the polynucleotide encodes a protein to be produced in high yield (as will often be the case, for example, when therapeutic proteins are to be expressed)
  • it will often be desirable to select plants with relatively high biomass e.g., tobacco, which has the additional advantages that it is highly susceptible to viral infection, has a short growth period, and is not in the human food chain).
  • polynucleotide encodes a protein whose full activity requires (or is inhibited by) a particular post-translational modification
  • the ability (or inability) of certain plant species to accomplish the relevant modification may direct selection.
  • crop plants, or crop-related plants are utilized.
  • edible plants are utilized.
  • Preferred plants for use in accordance with the present invention include Angiosperms, Bryophytes (e.g., Hepaticae, Musci, etc.), Pteridophytes (e.g., ferns, horsetails, lycopods), Gymnosperms (e.g., conifers, cycase, Ginlco, Gnetales), and Algae (e.g., Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, and Euglenophyceae).
  • Bryophytes e.g., Hepaticae, Musci, etc.
  • Pteridophytes e.g., ferns, horsetails, lycopods
  • Gymnosperms e.g., conifers, cycase, Ginlco, Gnetales
  • Algae e.g., Chlo
  • Fabaceae members ofthe family Leguminosae (Fabaceae; e.g., pea, alfalfa, soybean); Gramineae (Poaceae; e.g., corn, wheat, rice); Solanaceae, particularly ofthe genus Lycopersicon (e.g., tomato), Solanum (e.g., potato, eggplant), Capsium (e.e., pepper), or Nicotiana (e.g., tobacco); Umbelliferae, particularly ofthe genus Daucus (e.g., carrot), Apium (e.g., celery), or Rutaceae (e.g., oranges); Compositae, particularly ofthe genus Lactuca (e.g., lettuce); Brassicaceae (Cruciferae), particularly ofthe genus Brassica or Sinapis.
  • Brassicaceae family members include Brassica campesfris, B. carinata, B. juncea, B. napus, B. nigra, B. oleraceae,B. toumifortii, Sinapis alba, and Raphanus sativus.
  • polynucleotides and Polypeptides of Interest may be employed to deliver to and/or express in plant cells any polynucleotide of interest.
  • protein-coding polynucleotides may express enzymes, antibodies, hormones, cytokines, regulatory factors, structural proteins, or any other protein or polypeptide of interest.
  • Encoded proteins may be naturally-occurring proteins, or may be designed or engineered proteins, including for instance fusion proteins (e.g., fusion proteins incorporating part or all of a plant virus protein such as MP or CP). See, e.g., U.S. Pat. Nos. 6,448,070 and 6,660,500.
  • fusion proteins Numerous types may be encoded.
  • a heterologous sequence may be fused to the 5' or 3' end of a plant virus protein or located internally. Numerous sequences of diverse origin may be included within a single fusion protein.
  • the encoded protein may comprise a cleavage site, which may be encoded by the inserted polynucleotide or by the viral vector. See, e.g., U.S. Pat. No. 6,740,740.
  • the vector may comprise a portion that encodes a cleavage site upstream of a portion that encodes CP so that when a polynucleotide of interest is inserted between the CP promoter and the portion that encodes a cleavage site, the resulting open reading frame encodes a fusion protein containing a portion encoded by the polynucleotide of interest, a cleavage site, and part or all ofthe CP. Cleavage ofthe fusion protein at the cleavage site releases the encoded polypeptide of interest.
  • the cleavage site may be a site for cleavage by chemical means (e.g., cyanogen bromide) or by enzymatic means (e.g., by a protease such as trypsin, chymotrypsin, thrombin, pepsin, Staphylococcus aureus N8 protease, and Factor Xa protease).
  • a protease such as trypsin, chymotrypsin, thrombin, pepsin, Staphylococcus aureus N8 protease, and Factor Xa protease.
  • the polynucleotide of interest comprises a portion encoding a tag, e.g., a 6X-His tag, HA tag, Myc tag, FLAG tag, etc. Such tags may simplify the detection, isolation and/or purification ofthe protein.
  • the tag is a cleavable tag, e.g., a tag cleavable by chemical means or by enzymatic means as described above. Including a cleavage site allows the tag to be readily be removed from the translated polypeptide, e.g., after purification, resulting in a protein with wild type sequence. It is to be understood that the tag and/or cleavage site may be present within a viral vector into which a particular polynucleotide of interest is to be inserted and need not be present within the inserted polynucleotide itself. Once the polynucleotide is inserted, the entire portion comprising the region(s) that encode the tag, cleavage site, and newly inserted polynucleotide is considered a polynucleotide of interest.
  • inventive system to express more than one polypeptide chain in the same clonal root or plant cell line or clonal plant (e.g., using two different viral vectors each of which directs expression of a polynucleotide, inserting two different polynucleotides into one viral vector, utilizing a transgenic plant that expresses one or more polynucleotides to generate a clonal root or plant cell line or clonal plant), for example in order to produce a multimeric protein or to simultaneously produce two different proteins such as a protein of interest and a detectable or selectable marker).
  • a polynucleotide that encodes a therapeutically active protein is employed.
  • proteins that have been approved for therapeutic uses include, for example, insulin, human growth hormone, interferons, albumin, tPA, erythropoietm, interleukins, factor NIII, D ⁇ ase, factor IX, PDGF, FSH, T ⁇ F receptor (soluble form), calcitonin, and a variety of immunoglobulins.
  • the invention is not limited to such approved proteins, but encompasses expression of any polynucleotide(s), whether protein-coding or not, and particularly encompasses expression of any polynucleotide encoding any therapeutically active protein, whether prokaryotic or eukaryotic in origin, etc.
  • the pharmaceutical proteins of interest include, but are not limited to, hormones (insulin, thyroid hormone, catecholamines, gonadotrophines, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins and the like), growth hormones (e.g., human grown hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor and the like), growth factor receptors, cytokines and immune system proteins (e.g., interleukins, colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), granulocyte- macrophage colony stimulating factor (GM-CSF), erythropoietm, tumor necrosis factor (T ⁇ F), interfersons, integrins, addressins, seletins, homing receptors, T cell receptors, immunoglobulins, soluble major histocompatibility complex antigens, immunologically active antigen
  • the present invention may be utilized to produce vaccine components.
  • proteins or polypeptides that may be formulated in a vaccine include virtually any potentially antigenic protein or portion thereof, for example, viral coat proteins, viral fusion proteins, viral envelope proteins, viral glycoproteins, bacterial or fungal cell wall proteins, toxin proteins, parasite coat proteins, tumor-specific antigens, etc., or portions of any ofthe foregoing. See, e.g., WO9640229.
  • Viruses of interest include HIV, respiratory syncytial virus (RSV), rabies virus, polio virus, pneumoviruses, metapneumoviruses, influenza viruses, poxviruses (including smallpox), rhinoviruses, coronavimses, adenoviruses, herpesviruses, hantaviruses, Ebola virus, Yellow Fever virus, Dengue virus, hepatitis viruses (e.g., hepatitis A, B, C, D, E, F, or G virus) etc.
  • Bacteria of interest include Neisseria, Pneumococcus, Streptococcus, H. influenzae, Staphylococcus, anthrax, etc.
  • Parasites of interest include Plasmodium, Leishmania, Toxoplasma, Ascaris, hookworm and other nematodes, ameba, flukes, etc.
  • the inventive system may be used to express a polynucleotide encoding an . enzyme that synthesizes or modifies a biologically active agent.
  • an enzyme that synthesizes or modifies a biologically active agent.
  • certain enzymes e.g., polyketide synthases, polypeptide synthetases, terpene synthases, etc.
  • synthesize small molecules with interesting biological activities including therapeutic activities (e.g., antibiotic, anticancer, immunosuppressive activities, etc.).
  • therapeutic activities e.g., antibiotic, anticancer, immunosuppressive activities, etc.
  • a large number of enzymes that modify protein or small molecule substrates e.g., kinases, hydrolases, transferases, etc.
  • the polynucleotide encodes a component (e.g., an enzyme) in a biosynthetic pathway.
  • Plants are a source of numerous natural products of use for medicinal and/or industrial purposes and others. It is of interest to increase the level or efficiency by which such products are produced.
  • a polynucleotide of interest may encode a biosynthetic enzyme, e.g., an enzyme that catalyzes a rate-limiting step in a biosynthetic pathway, by which such natural product(s) are synthesized.
  • the inventive system may be used to produce diagnostic or research reagents including, for example, antibodies.
  • the polynucleotide encodes a protein that enhances plant growth or survival in any of a variety of ways.
  • the protein may enhance the ability ofthe plant to extract nutrients from soil or culture medium, may confer resistance to an environmental condition such as temperature, salinity, etc., or may confer resistance to a pathogen such as a virus, bacterium, fungus, nematode, insect, etc.
  • Such proteins include both endogenous plant proteins (i.e., proteins that are naturally expressed in the plant from which the clonal root line, clonal plant cell line, or clonal plant is derived) and non-endogenous proteins.
  • inventive system may be utilized to produce nutritionally relevant proteins or other products.
  • Nutritionally relevant proteins include, for example, proteins that are found naturally in foods consumed by humans or domesticated animals (e.g., cats, dogs).
  • Other examples include proteins having a balanced amino acid composition, e.g., proteins having an amino acid composition such as those used for total parenteral nutrition (TPN), etc.
  • the inventive system may be utilized to express polynucleotides that do not necessarily encode proteins, for example to produce active RNA species, e.g., ribozymes or interfering RNAs that silence gene expression (either long double-stranded RNAs or short interfering RNAs (siRNAs), microRNAs or microRNA precursors, short hairpin RNAs (shRNAs), etc. See, e.g., U.S. Pat. No. 6,531,647; 6,635,805 and U.S. Pub. No. 20040019930.
  • ribozymes or interfering RNAs may be produced that target plant genes, so that an altered plant is created, for example that does not express a particular receptor for a plant pathogen, or a particular allergenic protein.
  • V. Isolation and/or Formulation of Polynucleotide Expression Products it will be desirable to isolate polynucleotide expression products from the plant tissue(s), e.g., roots, root cells, plants, plant cells, that express them. It may also be desirable to formulate such isolated products for their intended use (e.g., as a pharmaceutical or diagnostic agent, or as a reagent, etc.). In other embodiments, it will be desirable to formulate the products together with some or all ofthe plant tissues that express them. [00136] Where it is desirable to isolate the expression product from some or all of the plant cells or tissues that express it, any available purification techniques may be employed.
  • the polynucleotide encodes a nutritionally relevant protein, or a therapeutic protein that is active after oral delivery (when properly formulated)
  • the polynucleotide encodes or produces a therapeutic agent
  • it may be formulated according to know techniques.
  • an effective amount of a pharmaceutically active product can be formulated together with one or more organic or inorganic, liquid or solid, pharmaceutically suitable carrier materials.
  • a pharmaceutically active product produced according to the present invention may be employed in dosage forms such as tablets, capsules, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, powder packets, liquid solutions, solvents, diluents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and solid bindings, as long as the biological activity ofthe protein is not destroyed by such dosage form.
  • Materials that can serve as pharmaceutically acceptable carriers include, but are not limited to sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium
  • the polynucleotide expression product may be provided as a pharmaceutical composition by means of conventional mixing granulating dragee-making, dissolving, lyophilizing, or similar processes.
  • the pharmaceutically active product e.g., protein
  • the rate of absorption ofthe product then depends upon its rate of dissolution, which in turn, may depend upon size and form.
  • delayed absorption of a parenterally administered product is accomplished by dissolving or suspending the product in an oil vehicle.
  • injectable depot forms are made by forming microcapsule matrices ofthe protein in biodegradable polymers such as polylactide-polyglycolide.
  • the rate of release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations may be prepared by entrapping the product in liposomes or microemulsions, which are compatible with body tissues.
  • Enterally administered preparations of pharmaceutically active products may be introduced in solid, semi-solid, suspension or emulsion form and may be compounded with any pharmaceutically acceptable carriers, such as water, suspending agents, and emulsifying agents.
  • the expression products may also be administered by means of pumps or sustained-release forms, especially when administered as a preventive measure, so as to prevent the development of disease in a subject or to ameliorate or delay an already established disease.
  • compositions for oral administration include plants; extractions ofthe plants, and proteins purified from infected plants provided as dry powders, foodstuffs, aqueous or non-aqueous solvents, suspensions, or emulsions.
  • non- aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil, fish oil, and injectable organic esters.
  • Aqueous carriers include water, water-alcohol solutions, emulsions or suspensions, including saline and buffered medial parenteral vehicles including sodium chloride solution, Ringer's dextrose solution, dextrose plus sodium chloride solution, Ringer's solution containing lactose or fixed oils.
  • dry powders include any plant biomass that has been dried, for example, freeze dried, air dried, or spray dried. For example, the plants may be air dried by placing them in a commercial air dryer at about 120 degrees Fahrenheit until the biomass contains less than 5% moisture by weight.
  • the dried plants may be stored for further processing as bulk solids or further processed by grinding to a desired mesh sized powder.
  • freeze-drying may be used for products that are sensitive to air-drying. Products may be freeze dried by placing them into a vacuum drier and dried frozen under a vacuum until the biomass contains less than about 5% moisture by weight.
  • the dried material can be further processed as described herein.
  • Plant-derived material may be administered as or together with one or more herbal preparations.
  • Useful herbal preparations include liquid and solid herbal preparations.
  • Some examples of herbal preparations include tinctures, extracts (e.g., aqueous extracts, alcohol extracts), decoctions, dried preparations (e.g., air-dried, spray dried, frozen, or freeze-dried), powders (e.g., lyophilized powder), and liquid.
  • Herbal preparations can be provided in any standard delivery vehicle, such as a capsule, tablet, suppository, liquid dosage, etc. Those skilled in the art will appreciate the various formulations and modalities of delivery of herbal preparations that may be applied to the present invention.
  • Plant material e.g., roots, leaves, etc.
  • Plants may also be extracted in a buffered solution.
  • the plant material may be transferred into an amount of ice-cold water at a ratio of one to one by weight that has been buffered with, e.g., phosphate buffer.
  • Protease inhibitors can also be added as required.
  • the plant material can be disrupted by vigorous blending or grinding while suspended in the buffer solution and the extracted biomass removed by filtration or centrifugation.
  • the product carried in solution can be further purified by additional steps or converted to a dry powder by freeze-drying or precipitation. Extraction can also be carried out by pressing. Plants or roots can also be extracted by pressing in a press or by being crushed as they are passed through closely spaced rollers. The fluids expressed from the crushed plants or roots are collected and processed according to methods well known in the art. Extraction by pressing allows the release ofthe products in a more concentrated form. However, the overall yield of the product may be lower than if the product were extracted in solution. [00145] Inventive root lines, cell lines, plants, extractions, powders, dried preparations and purified protein or nucleic acid products, etc., can also be in encapsulated form with or without one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active product may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents.
  • opacifying agents may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part ofthe intestinal tract, optionally, in a delayed manner.
  • embedding compositions include polymeric substances and waxes.
  • a plant or portion thereof expressing a pharmaceutically active product according to the present invention, or biomass thereof is administered orally as medicinal food.
  • Such edible compositions are consumed by eating raw, if in a solid form, or by drinking, if in liquid form.
  • the plant material is directly ingested without a prior processing step or after minimal culinary preparation.
  • the plant biomass is processed and the material recovered after the processing step is ingested.
  • Processing methods preferably used in the present invention are methods commonly used in the food or feed industry. The final products of such methods still include a substantial amount ofthe expressed pharmaceutically active polynucleotide or polypeptide and are preferably conveniently eaten or drun .
  • the final product may also be mixed with other food or feed forms, such as salts, carriers, favor enhancers, antibiotics, and the like, and consumed in solid, semi-solid, suspension, emulsion, or liquid form.
  • such methods include a conservation step, such as, e.g., pasteurization, cooking, or addition of conservation and preservation agents. Any plant is used and processed in the present invention to produce edible or drinkable plant matter.
  • the amount of pharmaceutically active polynucleotide or polypeptide expression product in a plant-derived preparation may be tested by methods standard in the art, e.g., gel electrophoresis, ELISA, or Western blot analysis, using a probe or antibody specific for the product.
  • This determination may be used to standardize the amount of polynucleotide or protein ingested.
  • the amount of therapeutically active product may be determined and regulated, for example, by mixing batches of product having different levels of product so that the quantity of material to be drunk or eaten to ingest a single dose can be standardized.
  • a pharmaceutically active polynucleotide or protein produced in a plant cell or tissue and eaten by a host is preferably absorbed by the digestive system.
  • One advantage ofthe ingestion of plant tissue that has been only minimally processed is to provide encapsulation or sequestration ofthe polynucleotide or protein in cells ofthe plant.
  • the product may receive at least some protection from digestion in the upper digestive tract before reaching the gut or intestine and a higher proportion of active product would be available for uptake.
  • compositions ofthe present invention can be administered therapeutically or prophylactically.
  • the compositions may be used to treat or prevent a disease.
  • any individual who suffers from a disease or who is at risk of developing a disease may be treated.
  • an individual can be considered at risk for developing a disease without having been diagnosed with any symptoms ofthe disease.
  • the individual has a particular genetic marker identified as being associated with increased risk for developing a particular disease, that individual will be considered at risk for developing the disease.
  • members of an individual's family have been diagnosed with a particular disease, e.g., cancer, the individual may be considered to be at risk for developing that disease.
  • Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetraliydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compositions of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active protein.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active protein.
  • Dosage forms for topical or transdermal administration of a pharmaceutical composition of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active product, or preparation thereof is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation, eardrops, and eye drops are also contemplated as being within the scope of this invention.
  • the present invention contemplates the use of transdermal patches, which have the added advantage of providing controlled delivery of a pharmaceutically active protein to the body.
  • Such dosage forms can be made by suspending or dispensing the pharmaceutically active product in the proper medium.
  • Absorption enhancers can also be used to increase the flux ofthe pharmaceutically active protein across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the pharmaceutically active protein in a polymer matrix or gel.
  • the compositions are administered in such amounts and for such time as is necessary to achieve the desired result.
  • a "therapeutically effective amount" of a pharmaceutical composition is that amount effective for treating, attenuating, or preventing a disease in a host.
  • the "amount effective to treat, attenuate, or prevent disease" refers to a nontoxic but sufficient amount ofthe pharmaceutical composition to treat, attenuate, or prevent disease in any host.
  • the "therapeutically effective amount” can be an amount to treat, attenuate, or prevent diabetes, growth hormone deficiency, etc.
  • the "therapeutically effective amount” can be an amount sufficient to cause an immune response in a subject, e.g., the production of antibodies that bind to a particular antigen.
  • the antibodies protect against or reduce the severity of infection or protect against a disease or condition that may result from exposure to the antigen.
  • the exact amount required will vary from subject to subject, depending on the species, age, and general condition ofthe subject, the stage ofthe disease, the particular pharmaceutical mixture, its mode of administration, and the like.
  • the infected plants ofthe invention and/or protein preparations thereof are preferably formulated in dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to a physically discrete unit of pharmaceutically active polynucleotide or polypeptide expression product appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions ofthe present invention is preferably decided by an attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient or organism may depend upon a vaiiety of factors including the disorder being treated and the severity ofthe disorder; the activity ofthe specific compound employed; the specific composition employed; the age, body weight, general health, sex ofthe patient, diet ofthe patient, pharmacokinetic condition ofthe patient, the time of administration, route of administration, and rate of excretion ofthe specific compound employed; the duration ofthe treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well Icnown in the medical arts,
  • compositions ofthe present invention can be employed in combination therapies, that is, the pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
  • the particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeutic effect to be achieved.
  • the therapies employed may achieve a desired effect for the same disorder (for example, an inventive compound may be administered concurrently with another anti-cancer agent), or they may achieve different effects.
  • Example 1 Construction of Recombinant Plant Virus Vectors
  • FIG. 1 shows a schematic diagram of a TMN-based vector, D4, that was engineered to accept insertion of a polynucleotide of interest (Sl ivprasad et al, Virology, 255(2):312-23, 1999), and illustrates insertion of various polynucleotides of interest into the vector.
  • D4 contains a deletion ofthe TMN coat protein (CP) coding sequences but retains the TMN CP subgenomic promoter and the TMN 3' untranslated region (UTR), as indicated on the figure.
  • the 126 and 183 IcD proteins are required for TMN replication.
  • the 30 IcD protein is movement protein (MP), used for cell-to-cell movement.
  • MP movement protein
  • D4 contains Pac I and Xho I sites downstream ofthe CP subgenomic promoter, providing a site for convenient insertion of a polynucleotide of interest. Particular vectors created by inserting various polynucleotides of interest into D4 are described below.
  • D4C3GFP is a TMN-based expression vector that is deficient in CP production (Sliivprasad et al, 1999: TTT-GFP) as a result of deletion ofthe TMN CP coding region and its replacement with the C3GFP gene, which is placed under the control ofthe TMN CP subgenomic promoter.
  • the C3GFP gene was recloned into D4 by overlapping PCR to eliminate the Ncol dXhol sites in the C3GFP nucleotide sequence to facilitate further cloning steps.
  • a poly linker Pstl-Notl-Xhol was introduced at the 3 'end of C3GFP gene.
  • the PCR product digested with Pacl-Xhol was cloned into D4 resulting in D4C3GFP.
  • Three constructs that contained full-length or portions ofthe 3 '-untranslated region (3' UTR) of AIMN R ⁇ A3 were also generated.
  • sequences encoding C3GFP under control ofthe subgenomic TMN CP promoter were present upstream of AIMN R ⁇ A3 3' -UTR sequences (either full-length or a portion of the UTR), to allow us to precisely identify the sequences ofthe AIMN R ⁇ A3 3' UTR required for assembly and movement of TMN genomic R ⁇ A (either in trans or in cis).
  • the R ⁇ A3 sequences were inserted between the Notl and- ⁇ 7zoI sites ofthe new D4C3GFP vector as Notl-Sall fragments, resulting in the constructs SR25 (nts 1859- 1941 of R ⁇ A3), SR26 (nts. 1859-1969 of RNA3) and SR27 (nts. 1859-2037 of RNA3, i.e., the entire 3' UTR) ( Figure lid).
  • sequences from the AIMV RNA3 3' UTR, SR25, SR26, and SR27 also include sequences from the TMV 3' UTR (i.e., the UTR from the TMV genomic transcript) downstream ofthe inserted AIMV sequences.
  • TMV nucleotides 6192-6395 are TMV nucleotides 6192-6395, as in the D4 construct.
  • the TMV-based viruses (SR25, SR26, and SR27) are defective in long-distance movement because the TMV coat protein is essential for effective phloem-mediated long distance transport and systemic infection of TMV.
  • Viral vectors in which polynucleotides of interest (e.g., GFP, hGH, GCSF) are inserted into SR25, SR26, and/or SR27 are in the process of being tested for generation of clonal root lines, clonal plant cell lines, and clonal plants as described herein.
  • Primer SR22 (5'-CCG TTAATTAATG TTC CCA ACT ATT CCA) was used to clone hGH without its leader, and introducing a Pad site at the 5' end; primer SR23 (5'-CCG TTAATTAATG GCA ACT GGA TCA AGG) was used to clone hGH with its leader.
  • Example 2 Generation and testing of clonal root lines expressing GFP
  • Materials and Methods [00167] Synthesis of viral transcripts and viral infection.
  • Agrobacterium rhizogenes stimulated root generation.
  • Agrobacterium rhizogenes strain A4RSII was grown to OD 600 0.8-1.
  • Bacterial cells were pelleted and resuspended in MS-2 medium (MS salts, 2% sucrose, lOmM MES, pH 5.5) to a final OD 6 oo of 0.5.
  • Acetosyringone was added to a final concentration 200 ⁇ M 1 hour before transformation.
  • Nicotiana benthamiana were harvested 5-14 days after inoculation with transcript. Leaves were surface sterilized for 6 min with 10%> Clorox and washed several times with sterile distilled water. [00169] Surface sterilized leaves of N. benthamiana were cut into pieces ⁇ 1 cm . They were dipped into bacterial suspension for 5 min, drained on filter paper and placed on the surface of solidified MS-2 medium. Plates were kept under dim light conditions at 24°C for 48 hours. After 48 hours the excess Agrobacterial suspension was removed, and leaf explants were placed on solid hormone free K 3 (Kao K. ⁇ .
  • Roots were screened for the presence ofthe protein of interest by Western blot analysis and/or by fluorescence under UN light, depending on the particular polynucleotide of interest.
  • benthamiana plants systemically infected with the same vector were harvested at the time of peak expression, and an extract was prepared in an identical manner as described above for the root material and loaded on the gel for comparison with the root cell lines.
  • electrophoresis proteins were electroblotted onto a nylon membrane, blocked using casein and reacted with GFP- specific antibodies (BD Biosciences Clontech). Proteins reacting with antibodies were visualized using a chemiluminescent substrate.
  • Figures 6A - 6E show the overall method used for generating the clonal root lines (see Description).
  • Figure 6G shows a photograph of a GFP-expressing clonal root line that was obtained by infecting N. benthamiana with viral vector D4C3GFP, harvesting leaf tissue from the infected region, infecting with A. rhizogenes, and culturing the pieces to allow development of hairy roots, which were then isolated and further cultured.
  • Figures 7A - 7C show Western blot analyses demonstrating GFP production in 3 clonal root lines derived from plant cells into which a viral vector whose genome contains a gene that encodes GFP under control ofthe TMN CP promoter (D4C3GFP) was introduced.
  • Figure 7 A shows GFP expression in the clonal root lines after 30 days of propagation in culture (i.e., 30 days after separation ofthe root from the leaf from which it was derived).
  • Figure 7B shows GFP expression in the clonal root lines after 60 days of propagation in culture (i.e., 60 days after separation ofthe root from the leaf from which it was derived).
  • C- represents control lanes containing no protein.
  • MWM represents molecular weight markers.
  • GFP-R represents samples from clonal root lines.
  • GFP-P represents GFP isolated from leaf tissue of a plant infected with the same construct used for generation ofthe clonal root lines.
  • Figure 7C is a control showing that the anti-GFP antibodies recognize commercially available GFP protein.
  • Figures 8 A and 8B show photographs of clonal root lines producing hGH (see Example 4) or GFP.
  • Figure 8 A shows a photograph of two clonal root lines taken under normal light conditions.
  • the plate on the left shows a clonal root line derived from a plant cell into which a viral vector whose genome contains a gene that encodes human growth hormone (hGH) under control ofthe TMN CP promoter was introduced.
  • the plate on the right shows a clonal root line derived from a plant cell into which a viral vector whose genome contains a gene that encodes green fluorescent protein (GFP) under control ofthe TMN CP promoter was introduced.
  • GFP green fluorescent protein
  • Figure 8B shows a photograph ofthe same clonal root lines as shown in Figure 8 A taken under UN light, demonstrating expression of GFP.
  • benthamiana plants were inoculated with a TMN-based vector, D4-hGH, containing an open reading frame encoding hGH under control ofthe TMV CP subgenomic promoter. Hairy roots were obtained and subcultured essentially as described in Example 2. Two weeks after separation from leaf discs, during the third round of subculture, the segments of roots were analyzed for hGH expression by Western blot assay ( Figure 9) essentially as described in Example 2. Five ng hGH protein (Research Diagnostics) was used as a control in all Western blots in which expression of hGH was tested. Anti-hGH antibodies were from Research Diagnostics. As can be seen from Figure 9, up to 80 % of the clonal root lines had detectable levels of hGH.
  • Figure 10 shows a Western blot, demonstrating that the clonal root lines maintained stable expression of hGH after 10 passages in which hGH expression in selected lines was several fold higher (250 ug/gram fresh root tissue) than that in leaves infected with the same virus construct (70 ug/gram fresh leaf tissue) when compared by Western blot.
  • Figures 8A and 8B show photographs of clonal root lines producing hGH and GFP.
  • Figure 8A shows a photograph of two clonal root lines taken under normal light conditions.
  • the plate on the left shows a clonal root line derived from a plant cell into which a viral vector whose genome contains a gene that encodes human growth hormone (hGH) under control ofthe TMV CP promoter was introduced.
  • the plate on the right shows a clonal root line derived from a plant cell into which a viral vector whose genome contains a gene that encodes green fluorescent protein (GFP) under control ofthe TMV CP promoter was introduced.
  • Figure 8B shows a photograph ofthe same clonal root lines as shown in Figure 8 A taken under UV light, demonstrating expression of GFP.
  • Example 4 Generation and testing of clonal root lines expressing GCSF
  • N. benthamiana plants were inoculated with a TMN-based vector, D4- GCSF, containing an open reading frame encoding GCSF under control ofthe TMN CP subgenomic promoter, and hairy roots were obtained essentially as described in Example 2.
  • Two weeks after separation from leaf discs the segments of roots were analyzed for GCSF expression by Western blot assay (Figure 11). As can be seen from Figure 11, up to 80 % ofthe clonal root lines had detectable levels of GCSF. We selected the highest producers and propagated them further.
  • FIG. 12 shows a Western blot, demonstrating that the clonal root lines maintained stable expression of GCSF after 10 passages (subculturmgs).
  • Five ng recombinant GCSF produced using an E. coli expression system was used as a control in all Westerns in which expression of GCSF was tested.
  • Anti-GCSF antibodies were from Oncogene Science. [00182]
  • Example 5 Generation and testing of clonal plant cell lines expressing GCSF
  • Cells were then taken to 30-50 ml with filter sterilized protoplasting solution: 0.4M mannitol, MES 20mM, pH5.5, Cellulase OnozukaRS (Yalcult Honsha Co.) 1%, Pectolyase Y23 (Seishin Pharmaceutical Co.) 0.1%). Cells were incubated in 250 ml flasks at 25°C for 20-25 min. The protoplast solution was filtered through a 100/ ⁇ m sieve, spun at 700 rpm for 6 min, and washed 2x with ice-cold 0.4M Mamiitol.
  • Protoplasts were counted using a hemacytometer and resuspended in electroporation buffer: lOmM HEPES, 150 mM NaCl. 5 mM CaCl 2 , 0.4M mannitol, pH 7.2 to a final concentration lxl 0 6 protoplasts/ml.
  • Transcript 25-30 ⁇ l was placed into an electroporation cuvette, 0.4 cm (Biorad) kept on ice, and after 10-15 min was mixed with 0.5 ml of protoplast suspension by Pasteur pipette and immediately used to electroporate cells. Electroporation was performed using a Biorad Gene Pulser at 250 volts and 175 capacitance.
  • Electroporated protoplasts were resuspended in 8 ml of PBS buffer containing .4 M mannitol and maintained for formation ofthe cell wall.
  • Enrichment for stable producer cell lines Within 4 - 5 days following electroporation, dividing cells were diluted and sampled (10 ul of infected cells into 100 ul of medium) to enrich for cells that expressed the polynucleotide of interest (target molecule) at high levels. The diluted cells were spotted onto individual sections of a Petri dish, as shown in Figure 13E.
  • each sample was tested by visual or other means (e.g., Western blot) for the presence of target molecule (e.g., GFP, GCSF, hGH, etc.). Stably infected cells producing target molecule were selected for further enrichment until producer cell line is obtained.
  • target molecule e.g., GFP, GCSF, hGH, etc.
  • Stably infected cells producing target molecule were selected for further enrichment until producer cell line is obtained.
  • Results Clonal plant cell lines were derived by introducing a TMV-based viral vector containing an open reading frame that encodes GCSF under control ofthe TMV CP subgenomic promoter into BY-2 cells. The overall process is shown in Figure 13. Enrichment for cells that express GCSF was performed using Western blot assays until populations of cells (either single clonal cell lines or populations containing several clonal cell lines) were obtained.
  • Figure 14 shows Western blot analyses demonstrating GCSF production in a plant cell population derived from plant cells into which a viral vector whose genome contains a gene that encodes GCSF under control ofthe TMV CP promoter was introduced. It is noted that the enriched plant cell population may contain either a single clonal cell line or multiple lines. Further enrichment, using more dilute samples, would result in clonal cell lines.
  • Figure 14A shows a Western blot performed 48 hours after introduction ofthe vector.
  • Figure 14B shows a Western blot performed using the same cell populations as shown in Figure 14A performed after further maintaining the cells in culture, i.e., 57 days after inoculation.
  • GCSF-COM indicates a lane in which recombinant GCSF protein was loaded as a positive control.
  • MWM indicates molecular weight markers.
  • C- indicates a lane in which plant extract made from plants not expressing GCSF was loaded.
  • Example 6 Generation and testing of clonal cell lines expressing GFP [00189] Results
  • Clonal plant cell lines were derived by introducing a TMV-based viral vector containing an open reading frame that encodes GFP under control ofthe TMV CP subgenomic promoter (D4C3GFP) into BY-2 cells essentially as described in Example 5. Enrichment for cells that express GFP was performed using a visual screen for fluorescence until populations of cells (either single clonal cell lines or populations containing several clonal cell lines) that stably express GFP were obtained.
  • Figure 13C shows a protoplast suspension containing cells into which the viral vector was introduced.
  • Figure 13E shows diluted samples from the suspension plated in individual droplets on Petri dishes.
  • Figure 13F shows the same Petri dishes as in Figure 13E under UV light.
  • the droplets may contain either a single clonal plant cell line or multiple clonal plant cell lines.
  • Single clonal plant cell lines i.e, populations derived from a single ancestral cell
  • Single clonal plant cell lines can be generated by further limiting dilution using standard methods for single cell cloning.
  • Figure 15 shows GFP production in plant cell lines derived from plant cells into which D4C3GFP.
  • Figure 15 A shows enrichment for plant cell lines that express GFP.
  • Figure 15B shows a callus obtained from a clonal plant cell line that contains a similar viral vector that does not encode GFP. The photographs were taken 3 months after the vector was introduced into the cells from which the clones in Figure 15A were derived. Both photographs were taken under UV light.
  • Example 7 Generation and testing of a clonal plant
  • Clonal root lines expressing hGH were obtained as described in Example 3.
  • Root cells were isolated by enzymatic digestion and cultured as described in Peres et al., Plant Cell, Tissue, and Organ Culture 65, 37-44, 2001, to generate clonal plants.
  • Figure 16A shows a plant that was obtained from a clonal root line.
  • a small leaf sample was used to inoculate a tobacco variety that is a sensitive host for formation of local lesions upon viral infection.

Abstract

La présente invention concerne des systèmes et des méthodes servant à générer des lignées de racine clonale, des lignées cellulaires de racine clonale, et des plantes clonales et à exprimer des produits géniques dans ces lignées cellulaires et ces plantes. Selon certaines des méthodes de l'invention, un vecteur viral contenant un polynucléotide d'intérêt lié fonctionnellement à un promoteur est introduit dans une plante ou une partie associée. De la matière végétale (par exemple, des feuilles) renfermant le vecteur viral est utilisée pour engendrer des lignées de racine clonale, par exemple, par l'intermédiaire de leur contact avec A. rhizogenes en vue de former des racines chevelues. Ces dernières sont dérivées de cellules ancestrales uniques et sont donc clonales. Ces lignées de racine clonale contiennent le vecteur viral et expriment le polynucléotide d'intérêt. Des lignées cellulaires de racine clonale, des lignées cellulaires de plante clonale et des plantes clonales sont obtenues à partir des racines clonales. Selon d'autres méthodes de ladite invention, un vecteur viral contenant un polynucléotide d'intérêt lié fonctionnellement à un promoteur est introduit dans des cellules d'une lignée cellulaire végétale (par exemple, préexistante ou nouvellement dérivée) qui est maintenue en culture. Des lignées cellulaires de plante clonale qui expriment le polynucléotide d'intérêt sont obtenues, par exemple, par cycles successifs d'enrichissement, jusqu'à ce qu'une population de cellules présentant une expression stable soit obtenue. Des plantes clonales, dont des cellules renferment le vecteur viral sont obtenues à partir des lignées cellulaires de plante clonale, au moyen des méthodes traditionnelles. Ladite invention a aussi pour objet des lignées de racine clonale, des lignées cellulaires de racine clonale, des lignées cellulaires de plante clonale et des plantes clonales engendrées à l'aide des méthodes susmentionnées.
PCT/US2005/005409 2004-02-20 2005-02-18 Systemes et methodes d'expression clonale dans des plantes WO2005081905A2 (fr)

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AU2004272972A1 (en) 2003-05-22 2005-03-24 Fraunhofer Usa, Inc. Recombinant carrier molecule for expression, delivery and purification of target polypeptides
EP1686176A1 (fr) * 2005-01-28 2006-08-02 Icon Genetics AG Production d'anticorps dans des plantes avec des vecteurs de virus à ARN à sense positif
KR101446025B1 (ko) 2005-08-03 2014-10-01 아이바이오, 인크. 면역글로불린의 생산을 위한 조성물 및 방법
WO2009009759A2 (fr) 2007-07-11 2009-01-15 Fraunhofer Usa, Inc. Antigènes yersinia pestis, compositions de vaccins, et méthodes associées
WO2010037046A1 (fr) 2008-09-28 2010-04-01 Fraunhofer Usa, Inc. Anticorps anti-neuraminidase humanisé et procédés d’utilisation de celui-ci
EP4116316A1 (fr) 2015-07-04 2023-01-11 Evaxion Biotech A/S Protéines et acides nucléiques utiles dans des vaccins ciblant pseudomonas aeruginosa
WO2017216384A1 (fr) 2016-06-17 2017-12-21 Evaxion Biotech Aps Vaccination ciblant ichthyophthirius multifiliis
WO2017220787A1 (fr) 2016-06-24 2017-12-28 Evaxion Biotech Aps Vaccin contre l'infection provoquée par aeromonas salmonicida
EP3487872A1 (fr) 2016-07-22 2019-05-29 Evaxion Biotech ApS Protéines chimériques pour induire une immunité vis-à-vis d'une infection à s. aureus
WO2019145399A1 (fr) 2018-01-24 2019-08-01 Evaxion Biotech Aps Vaccins pour la prophylaxie d'infections par s. aureus
CN108913716A (zh) * 2018-08-01 2018-11-30 成都大学 一种快速诱导藜麦毛状根的方法
WO2020083904A1 (fr) 2018-10-22 2020-04-30 Evaxion Biotech Aps Vaccins ciblant m. catharrhalis
US20220143168A1 (en) 2019-02-27 2022-05-12 Evaxion Biotech A/S Vaccines targeting H. influenzae
CN112205512A (zh) * 2019-07-11 2021-01-12 江南大学 一种低致敏性植物蛋白肉的制备方法
WO2021140123A1 (fr) 2020-01-06 2021-07-15 Evaxion Biotech Aps Vaccins ciblant neisseria gonorrhoeae
CA3224564A1 (fr) 2021-07-05 2023-01-12 Andreas Holm MATTSSON Vaccins ciblant neisseria gonorrhoeae
WO2023148398A1 (fr) 2022-02-07 2023-08-10 Var2 Pharmaceuticals Aps Anticorps et fragments d'anticorps et analogues specifiques du sulfate de chondroïtine
WO2023213393A1 (fr) 2022-05-04 2023-11-09 Evaxion Biotech A/S Variants de protéine staphylococcique et parties tronquées

Family Cites Families (98)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3747501A (en) * 1966-04-19 1973-07-24 O Honda System and method for climate control in greenhouses
US4028847A (en) * 1976-02-19 1977-06-14 General Mills, Inc. Apparatus for producing plants
US4196265A (en) * 1977-06-15 1980-04-01 The Wistar Institute Method of producing antibodies
CA1192510A (fr) 1981-05-27 1985-08-27 Lawrence E. Pelcher Vecteur de arn virale de virus des plantes ou portion de ce vecteur, methode de construction, et methode de production d'un gene derive
NL8300698A (nl) * 1983-02-24 1984-09-17 Univ Leiden Werkwijze voor het inbouwen van vreemd dna in het genoom van tweezaadlobbige planten; agrobacterium tumefaciens bacterien en werkwijze voor het produceren daarvan; planten en plantecellen met gewijzigde genetische eigenschappen; werkwijze voor het bereiden van chemische en/of farmaceutische produkten.
US4816567A (en) * 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US5447858A (en) * 1984-04-13 1995-09-05 Mycogen Plant Sciences, Inc. Heat shock promoter and gene
JPS61134325A (ja) * 1984-12-04 1986-06-21 Teijin Ltd ハイブリツド抗体遺伝子の発現方法
CA1288073C (fr) * 1985-03-07 1991-08-27 Paul G. Ahlquist Vecteur de transformation de l'arn
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4956282A (en) 1985-07-29 1990-09-11 Calgene, Inc. Mammalian peptide expression in plant cells
US5618920A (en) * 1985-11-01 1997-04-08 Xoma Corporation Modular assembly of antibody genes, antibodies prepared thereby and use
US5175102A (en) 1986-01-23 1992-12-29 Agricultural Genetics Company, Limited Modification of plant viruses or their effects
US5750871A (en) * 1986-05-29 1998-05-12 Calgene, Inc. Transformation and foreign gene expression in Brassica species
US5565347A (en) * 1986-06-10 1996-10-15 Calgene, Inc. Transformation and foreign gene expression with plant species
DE3850683T2 (de) * 1987-02-09 1994-10-27 Lubrizol Genetics Inc Hybrides RNS-Virus.
US6054566A (en) * 1988-02-26 2000-04-25 Biosource Technologies, Inc. Recombinant animal viral nucleic acids
US6846968B1 (en) * 1988-02-26 2005-01-25 Large Scale Biology Corporation Production of lysosomal enzymes in plants by transient expression
US5316931A (en) * 1988-02-26 1994-05-31 Biosource Genetics Corp. Plant viral vectors having heterologous subgenomic promoters for systemic expression of foreign genes
US6660500B2 (en) 1988-02-26 2003-12-09 Large Scale Biology Corporation Production of peptides in plants as viral coat protein fusions
US5922602A (en) 1988-02-26 1999-07-13 Biosource Technologies, Inc. Cytoplasmic inhibition of gene expression
US5223409A (en) * 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
CA1339307C (fr) 1988-09-06 1997-08-19 Roy Curtiss, Iii Immunisation orale par des plantes transgeniques
GB8823869D0 (en) * 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
US5530101A (en) * 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US7005560B1 (en) * 1989-10-27 2006-02-28 The Scripps Research Institute Transgenic plants expressing assembled secretory antibodies
US5427908A (en) * 1990-05-01 1995-06-27 Affymax Technologies N.V. Recombinant library screening methods
US5633425A (en) * 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5545806A (en) * 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5661016A (en) * 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
ES2246502T3 (es) * 1990-08-29 2006-02-16 Genpharm International, Inc. Animales no humanos transgenicos capaces de producir anticuerpos heterologos.
US5625126A (en) * 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
US5770429A (en) * 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5698426A (en) * 1990-09-28 1997-12-16 Ixsys, Incorporated Surface expression libraries of heteromeric receptors
US6288304B1 (en) * 1991-02-22 2001-09-11 Sembiosys Genetics Inc. Expression of somatotropin in plant seeds
CA2082160C (fr) * 1991-03-06 2003-05-06 Mary M. Bendig Anticorps monoclonaux humanises et chimeriques
DE69233367T2 (de) * 1991-04-10 2005-05-25 The Scripps Research Institute, La Jolla Bibliotheken heterodimerer rezeptoren mittels phagemiden
GB9108386D0 (en) 1991-04-19 1991-06-05 Agricultural Genetics Co Modified plant viruses as vectors
CA2078539C (fr) * 1991-09-18 2005-08-02 Kenya Shitara Procede de fabrication de chimere d'anticorps humain
US5565332A (en) * 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
GB9125979D0 (en) * 1991-12-06 1992-02-05 Wellcome Found Antibody
US5667988A (en) * 1992-01-27 1997-09-16 The Scripps Research Institute Methods for producing antibody libraries using universal or randomized immunoglobulin light chains
ATE171472T1 (de) * 1992-02-19 1998-10-15 Schering Corp Klonierung und expression von humanisierten monoklonalen antikörpern gegen menschliches interleukin-4
US6093399A (en) * 1992-03-05 2000-07-25 Board Of Regents, The University Of Texas System Methods and compositions for the specific coagulation of vasculature
US5965132A (en) * 1992-03-05 1999-10-12 Board Of Regents, The University Of Texas System Methods and compositions for targeting the vasculature of solid tumors
US6004555A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for the specific coagulation of vasculature
US6004554A (en) * 1992-03-05 1999-12-21 Board Of Regents, The University Of Texas System Methods for targeting the vasculature of solid tumors
US5877289A (en) * 1992-03-05 1999-03-02 The Scripps Research Institute Tissue factor compositions and ligands for the specific coagulation of vasculature
US5733743A (en) * 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
EP1162267A3 (fr) * 1992-03-31 2002-12-18 Kanebo Limited Vecteur de virus végétal, plasmid, procédé d'expression d'un gène étranger, et procédé de récupération de produit de gène étranger
US5639641A (en) * 1992-09-09 1997-06-17 Immunogen Inc. Resurfacing of rodent antibodies
IL108241A (en) * 1992-12-30 2000-08-13 Biosource Genetics Corp Plant expression system comprising a defective tobamovirus replicon integrated into the plant chromosome and a helper virus
DE69332763T2 (de) * 1992-12-30 2004-03-18 Biosource Genetics Corp., Vacaville Virale amplifikation rekombinanter boten-rna in transgenen pflanzen
US5491076A (en) * 1993-11-01 1996-02-13 The Texas A&M University System Expression of foreign genes using a replicating polyprotein producing virus vector
US5693506A (en) * 1993-11-16 1997-12-02 The Regents Of The University Of California Process for protein production in plants
US5756671A (en) 1994-06-02 1998-05-26 Mitotix, Inc. CDC37 cell-cycle regulatory protein, and uses related thereto
US5705154A (en) * 1995-03-08 1998-01-06 Schering Corporation Humanized monoclonal antibodies against human interleukin-4
US5702892A (en) * 1995-05-09 1997-12-30 The United States Of America As Represented By The Department Of Health And Human Services Phage-display of immunoglobulin heavy chain libraries
US6165718A (en) 1996-01-10 2000-12-26 Novo Nordisk A/S Novo Alle Method for in vivo production of a mutant library in cells
US5728300A (en) * 1996-02-15 1998-03-17 Phytotech, Inc. Phytorecovery of metals using seedlings
US5917117A (en) * 1996-03-21 1999-06-29 Phytotech, Inc. Inducing hyperaccumulation of metals in plant shoots
US6042832A (en) 1996-08-28 2000-03-28 Thomas Jefferson University Polypeptides fused with alfalfa mosaic virus or ilarvirus capsid proteins
CA2267078A1 (fr) 1996-10-04 1998-04-09 University Of Georgia Research Foundation Inc. Lichenase fongique et sequences codantes
US6127145A (en) * 1997-02-13 2000-10-03 Applied Phytologics, Inc. Production of α1 -antitrypsin in plants
GB9703146D0 (en) 1997-02-14 1997-04-02 Innes John Centre Innov Ltd Methods and means for gene silencing in transgenic plants
US5939541A (en) * 1997-03-28 1999-08-17 University Of South Carolina Method for enhancing expression of a foreign or endogenous gene product in plants
ZA984232B (en) * 1997-05-20 1998-12-01 Cornell Res Foundation Inc Grapevine leafroll virus type 2 proteins coding sequences and methods
US6297357B1 (en) * 1997-08-21 2001-10-02 Thomas Jefferson University prb2/p130 peptide inhibitors of cdk2 kinase activity
GB9720148D0 (en) 1997-09-22 1997-11-26 Innes John Centre Innov Ltd Gene silencing materials and methods
US6051239A (en) * 1997-10-20 2000-04-18 Thomas Jefferson University Compositions and methods for systemic delivery of oral vaccines and therapeutic agents
US6077992A (en) * 1997-10-24 2000-06-20 E. I. Du Pont De Nemours And Company Binary viral expression system in plants
US6632980B1 (en) * 1997-10-24 2003-10-14 E. I. Du Pont De Nemours And Company Binary viral expression system in plants
US20030211568A1 (en) 1997-10-27 2003-11-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US6700040B2 (en) * 1998-01-16 2004-03-02 Large Scale Biology Corporation Cytoplasmic gene inhibition or gene expression in transfected plants by a tobraviral vector
US6037456A (en) 1998-03-10 2000-03-14 Biosource Technologies, Inc. Process for isolating and purifying viruses, soluble proteins and peptides from plant sources
US6399317B1 (en) 1998-04-15 2002-06-04 Utah State University Real time detection of antigens
BR9912875A (pt) 1998-08-11 2002-02-13 Large Scale Biology Corp Método para a recuperação de proteìnas a partir do fluido intersticial de tecidos vegetais
EP1119581B1 (fr) * 1998-09-11 2005-12-21 Thomas Jefferson University UTILISATION D'UN VECTEUR EXPRIMANT RB2/p130 POUR EMPECHER OU TRAITER LA RESTENOSE OU LES PLAQUES D'ATHEROSCLEROSE
US6395962B1 (en) * 1999-06-22 2002-05-28 University Of South Carolina Enhancing expression of a silenced target sequence in plants using plant viral enhancers and amplicons
US6410317B1 (en) 1999-07-14 2002-06-25 Clontech Laboratories, Inc. Recombinase-based methods for producing expression vectors and compositions for use in practicing the same
NZ518947A (en) * 1999-11-22 2004-05-28 Plant Bioscience Ltd Enhanced expression through the use of a posttranscriptional gene silencing suppressor protein
FI19992659A (fi) * 1999-12-10 2001-06-11 Unicrop Ltd Prosessi kaksisirkkaisten siementen varastoravintoaineiden muuttamiseksi yhden tai useamman geenituotteen sisältäviksi aineosiksi
US6369296B1 (en) * 2000-02-01 2002-04-09 Plant Bioscience Limited Recombinant plant viral vectors
DE10109354A1 (de) * 2001-02-27 2002-09-05 Icon Genetics Ag Rekombinante virale Schaltersysteme
WO2003052108A2 (fr) * 2001-12-18 2003-06-26 Bayer Bioscience N.V. Methodes ameliorees et dispositif d'administration d'arn inhibiteur a des vegetaux et applications associees
US20040092470A1 (en) * 2002-06-18 2004-05-13 Leonard Sherry A. Dry powder oligonucleotide formualtion, preparation and its uses
US7012172B2 (en) * 2002-07-25 2006-03-14 Fraunhofer, Usa, Inc. Virus induced gene silencing in plants
US7326665B2 (en) * 2002-09-04 2008-02-05 Asahi Glass Company, Limited Light blue flat glass
EP1629090B1 (fr) * 2002-11-06 2014-03-05 iBio, Inc. Expression de sequences etrangeres dans des vegetaux utilisant un systeme de transactivation
US20070178148A1 (en) * 2002-11-12 2007-08-02 Fraunhofer U.S.A. Inc. Preparations of growth hormone
US8148608B2 (en) 2004-02-20 2012-04-03 Fraunhofer Usa, Inc Systems and methods for clonal expression in plants
US7683238B2 (en) * 2002-11-12 2010-03-23 iBio, Inc. and Fraunhofer USA, Inc. Production of pharmaceutically active proteins in sprouted seedlings
US7692063B2 (en) * 2002-11-12 2010-04-06 Ibio, Inc. Production of foreign nucleic acids and polypeptides in sprout systems
EP1594956A4 (fr) * 2003-02-03 2007-08-01 Fraunhofer Usa Inc Systeme d'expression de genes dans des plantes
AU2004272972A1 (en) * 2003-05-22 2005-03-24 Fraunhofer Usa, Inc. Recombinant carrier molecule for expression, delivery and purification of target polypeptides
EP1564295A1 (fr) * 2004-01-23 2005-08-17 Icon Genetics AG Système d'expression derivé des virus à ARN
WO2007135480A1 (fr) 2006-05-22 2007-11-29 Plant Bioscience Limited Système bipartite, procédé et composition permettant l'expression constitutive et inductible de niveaux élevés de protéines étrangères dans des plantes
AU2007267359B2 (en) 2006-05-29 2013-03-14 Icon Genetics Gmbh Plant virus-based inducible expression system

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None
See also references of EP1769068A4

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9765349B2 (en) 2003-02-03 2017-09-19 Ibio, Inc. System for expression of genes in plants
EP1984509A2 (fr) * 2006-02-13 2008-10-29 Fraunhofer USA, Inc. Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux
EP1984388A2 (fr) * 2006-02-13 2008-10-29 Fraunhofer USA, Inc. Antigenes du papillomavirus humain, compositions de vaccin et methodes
EP1984509A4 (fr) * 2006-02-13 2010-07-14 Fraunhofer Usa Inc Production d'acides nucleiques et de polypeptides etrangers dans des systemes vegetaux
EP1984388A4 (fr) * 2006-02-13 2012-08-01 Fraunhofer Usa Inc Antigenes du papillomavirus humain, compositions de vaccin et methodes
KR100791090B1 (ko) 2007-01-05 2008-01-04 대한민국 플라스미노겐 액티베이터 유전자를 포함하는 식물 형질전환용 벡터 및 상기 벡터에 의해 형질전환된 식물
KR100887367B1 (ko) 2007-07-12 2009-03-06 대한민국(관리부서:농촌진흥청) 플라스미노겐 액티베이터 유전자를 포함하는 벡터, 상기벡터에 의해 형질전환된 모상근 및 이를 이용한플라스미노겐 액티베이터의 제조방법
WO2011041391A1 (fr) 2009-09-29 2011-04-07 Fraunhofer Usa, Inc. Anticorps dirigés contre l'hémagglutinine du virus de la grippe, compositions, et procédés associés
US9809644B2 (en) 2009-09-29 2017-11-07 Ibio Inc. Influenza hemagglutinin antibodies, compositions and related methods
US11718648B2 (en) 2017-01-05 2023-08-08 Evaxion Biotech A/S Vaccines targeting Pseudomonas aeruginosa
WO2021123122A1 (fr) * 2019-12-18 2021-06-24 Genethon Production de vecteurs viraux recombinés à partir de racines velues de plantes

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EP1769068B1 (fr) 2014-12-31
EP1769068A2 (fr) 2007-04-04
ES2532609T3 (es) 2015-03-30
CN1922306B (zh) 2010-10-06
US20060085871A1 (en) 2006-04-20
WO2005081905A3 (fr) 2006-06-22
CA2555230A1 (fr) 2005-09-09
US8148608B2 (en) 2012-04-03
CN1922306A (zh) 2007-02-28
EP1769068A4 (fr) 2010-08-18
AU2005216133A1 (en) 2005-09-09

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